The effects of crocin on spatial memory impairment induced by hyoscine: Role of NMDA, AMPA, ERK, and CaMKII proteins in rat hippocampus

Document Type : Original Article


1 Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

3 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Shiraz University of Medical Sciences Shiraz Iran


Objective(s): Crocus sativus L. and its active constituent, crocin, have neuroprotective effects. The effects of crocin on memory impairment have been mentioned in studies but the signaling pathways  have not been evaluated. Therefore, the aim of this study was to evaluate the effects of crocin on the hyoscine-induced memory impairment in rat. Additionally, the level of NMDA (N-methyl-D-aspartate receptors), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicd acid), ERK (extracellular signal-regulated kinases), CaMKII (calcium (Ca2+)/calmodulin (CaM)-dependent kinaseII) mRNA and proteins were determined in rat hippocampus.
Materials and Methods: Crocin (10, 20, and 40 mg/kg), hyoscine (1.5 mg/kg), normal saline and rivastigmine  were administered intraperitoneally to male Wistar rats for 5 days. The effects on memory improvement were studied using Morris water maze (MWM) test. Then, the protein levels of NMDA, AMPA, ERK, pERK, CaMKII and p.CaMKII  in hippocampus were analized using the Western blot test. Furthermore, the mRNA levels of NMDA, AMPA, ERK and pCaMKII genes were evaluated using real-time quantitative reverse transcription-polymerase chain reaction (qRT- PCR) method.
Results: Aadminestration of crocin (20 mg/kg) and rivastigmine  significantly improved learning and memory impairment induced by hyoscine. Also, administration of hyoscine reduced  protein level of pERK,  while treatment with crocin (20 mg/kg) recovered the protein level.  No changes were observed in the protein levels and mRNA gene expression of NMDA, AMPA, ERK, CaMKII and pCaMKII following adminestration of hyoscine or crocin.
Conclusion: Adminestration of crocin improved memory and learning. The effect of crocin in this model can be mediated by alteration in pERK protein level in rat hippocampus.


Main Subjects

1. Alberini CM. Transcription factors in long-term memory and synaptic plasticity. Physiol Rev 2009; 89:121-145.
2. Giovannini MG, Lana D, Pepeu G. The integrated role of ACh, ERK and mTOR in the mechanisms of hippocampal inhibitory avoidance memory. Neurobiol Learn Mem 2015; 119:18-33.
3. McGaugh JL. Time-dependent processes in memory storage. Science 1966; 153:1351-1358.
4. Izquierdo I, Barros DM, Souza TM, de Souza MM, Izquierdo LA, Medina JH. Mechanisms for memory types differ. Nature 1998; 393:635-636.
5. Hyun Yi J, Hye Jin P, Ji Beak S, Lee S, Wook Jung J, Kim BC, et al. Danggui-Jakyak-San enhances hippocampal long-term potentiation through the ERK/CREB/BDNF cascade. J Intercult Ethnopharmacol 2015; 175:481-489.
6. Selkoe DJ. Alzheimer’s disease is a synaptic failure. Science  2002; 298:789-791.
7. Whitlock JR, Heynen AJ, Shuler MG, Bear MF. Learning induces long-term potentiation in the hippocampus. Science 2006; 313:1093-1097.
8. Henley JM, Wilkinson KA. AMPA receptor trafficking and the mechanisms underlying synaptic plasticity and cognitive aging. Dialogues Clin Neurosci 2013; 15:11-27.
9. Costa-Mattioli M, Sossin WS, Klann E, Sonenberg N. Translational control of long-lasting synaptic plasticity and memory. Neuron 2009; 61:10-26.
10. Tully T, Preat T, Boynton S, Del Vecchio M. Genetic dissection of consolidated memory in Drosophila. Cell 1994; 79:35-47.
11. Jarome TJ, Helmstetter FJ. Protein degradation and protein synthesis in long-term memory formation. Front Mol Neurosci 2014; 7:61. doi: 10.3389/fnmol.2014.00061.
12. McEntee WJ, Crook TH. Glutamate: its role in learning, memory, and the aging brain. Psychopharmacol 1993; 111:391-401.
13. Parent MB, Baxter MG. Septohippocampal Acetylcholine: Involved in but not necessary for learning and memory? Learn Mem 2004; 11:9-20.
14. Wang H, Peng R-Y. Basic roles of key molecules connected with NMDAR signaling pathway on regulating learning and memory and synaptic plasticity. Mil Med Res 2016; 3:26. doi:  10.1186/s40779-016-0095-0.
15.Grienberger C, Konnerth A. Imaging calcium in neurons. Neuron 2012; 73:862-885.
16. Nevian T, Sakmann B. Spine Ca2+ signaling in spike-timing-dependent plasticity. J Neurosci 2006; 26:11001-11013.
17. Bloodgood BL, Sabatini BL. Nonlinear regulation of unitary synaptic signals by CaV 2.3 voltage-sensitive calcium channels located in dendritic spines. Neuron 2007; 53:249-260.
18. Kim E, Choi E-J. Compromised MAPK signaling in human diseases: an update. Arch Toxicol  2015; 89:867-882.
19. Gutkind JS. The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem 1998; 273:1839-1842.
20. Picciotto MR, Higley MJ, Mineur YS. Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous system function and behavior. Neuron 2012; 76:116-129.
21. Francis PT. The interplay of neurotransmitters in Alzheimer’s disease. CNS Spectr 2005; 10:6-9.
22. Howes M-JR, Houghton PJ. Plants used in chinese and indian traditional medicine for improvement of memory and cognitive function. Pharmacol Biochem Behav 2003; 75:513-527.
23. Abd-Elhady RM, Elsheikh AM, Khalifa AE. Anti-amnestic properties of Ginkgo biloba extract on impaired memory function induced by aluminum in rats. Int J Dev Neurosci 2013; 31:598-607.
24. Rai GS, Shovlin C, Wesnes KA. ‘A double-blind, placebo controlled study of Ginkgo biloba extract (‘tanakan’) in elderly outpatients  with mild to moderate memory impairment’. Curr Med Res Opin 1991; 12: 350-355.
25. Hasanein P, Shahidi S. Effects of Hypericum perforatum extract on diabetes-induced learning and memory impairment in rats. Phytother Res  2011; 25: 544-549.
26. Wesnes KA, Ward T, McGinty A, Petrini O. ‘The memory enhancing effects of a Ginkgo biloba/Panax ginseng combination in healthy middle-aged volunteers’. Psychopharmacol 2000; 152: 353-361.
27. Javadi B, Sahebkar A, Emami SA. A Survey on saffron in major islamic traditional medicine books. Iran J Basic Med Sci 2013; 16:1-11.
28. Bathaie, Mousavi SZnA, Zeinab S. New applications and mechanisms of action of saffron and its important ingredients. Crit Rev Food Sci Nutr 2010; 50: 761-786.
29. Hosseinzadeh H, Nassiri-Asl M. Avicenna’s (Ibn Sina) the canon of medicine and saffron
( Crocus sativus ): A Review. Phytother Res 2013; 27: 475-483.
30. Rezaee Khorasany A, Hosseinzadeh H. Therapeutic effects of saffron (Crocus sativus L.) in digestive disorders: a review. Iran J Basic Med Sci 2016; 19:455-469.
31. Bolhasani A, Bathaie SZ, Yavari I, Moosavi-Movahedi A, Ghaffari SM. Separation and purification of some components of Iranian saffron. Asian J Chem 2005; 17: 725-729.
32. Sapanidou V, Taitzoglou I, Tsakmakidis Ι, Kourtzelis I, Fletouris D, Theodoridis A, et al. Antioxidant effect of crocin on bovine sperm quality and in vitro fertilization. Theriogenology 2015; 84:1273-1282.
33. Hosseinzadeh H, Shamsaie F, Mehri S. Antioxidant activity of aqueous and ethanolic extracts of Crocus sativus L stigma and its bioactive constituents crocin and safranal. Pharmacogn Mag 2010; 5: 419-424.
34. Sun Y, Xu H-J, Zhao Y-X, Wang L-Z, Sun L-R, Wang Z, et al. Crocin exhibits antitumor effects on human leukemia HL-60 cells in vitro and in vivo. Evid Based Complement Altern Med 2013; 2013: 1-7.
35. Noureini SK, Wink M. Antiproliferative effects of crocin in HepG2 cells by telomerase inhibition and hTERT down-regulation. Asian Pac J Cancer Prev 2012; 13:2305-2309.
36. Nam KN, Park Y-M, Jung H-J, Lee JY, Min BD, Park S-U, et al. Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells. Eur J Pharmacol 2010; 648:110-116.
37.Talaei A, Hassanpour Moghadam M, Sajadi Tabassi SA, Mohajeri SA. Crocin, the main active saffron constituent, as an adjunctive treatment in major depressive disorder: A randomized, double-blind, placebo-controlled, pilot clinical trial. J Affect Disord 2015; 174:51-56.
38. Vahdati Hassani F, Naseri V, Razavi BM, Mehri S, Abnous K, Hosseinzadeh H. Antidepressant effects of crocin and its effects on transcript and protein levels of CREB, BDNF, and VGF in rat hippocampus. Daru 2014; 22:16. doi: 10.1186/2008-2231-22-16.
39. Alavizadeh SH, Hosseinzadeh H. Bioactivity assessment and toxicity of crocin: a comprehensive review. Food Chem Toxicol 2014; 64:65-80.
40. Dorri SA, Hosseinzadeh H, Abnous K, Hasani FV, Robati RY, Razavi BM. Involvement of brain-derived neurotrophic factor (BDNF) on malathion induced depressive-like behavior in subacute exposure and protective effects of crocin. Iran J Basic Med Sci 2015; 18:958-966.
41. Tamaddonfard E, Gooshchi NH, Seiednejad-Yamchi S. Central effect of crocin on penicillin-induced epileptiform activity in rats. Pharmacol Rep 2012; 64:94-101.
42. Mollazadeh H, Emami SA, Hosseinzadeh H. Razi’s Al-Hawi and saffron (Crocus sativus): a review. Iran J Basic Med Sci 2015; 18:1153-1166.
43. Hosseinzadeh H, Ziaee T, Sadeghi A. The effect of saffron, Crocus sativus stigma, extract and its constituents, safranal and crocin on sexual behaviors in normal male rats. Phytomed 2008; 15:491-495.
44. Ghaeni FA, Amin B, Hariri AT, Meybodi NT, Hosseinzadeh H. Antilithiatic effects of crocin on ethylene glycol-induced lithiasis in rats. Urolithiasis 2014; 42:549-558.
45. Kianbakht S, Dabaghian F. Anti-obesity and anorectic effects of saffron and its constituent crocin in obese Wistar rat.  J Med Plants 2015; 14: 25-33.
46. Hosseinzadeh H, Abootorabi A, Sadeghnia HR. Protective effect of Crocus sativus stigma extract and crocin (trans-crocin 4) on methyl methanesulfonate-induced DNA damage in mice organs. DNA Cell Biol 2008; 27:657-664.
47. Ahmadi M, Rajaei Z, Hadjzadeh MA, Nemati H, Hosseini M. Crocin improves spatial learning and memory deficits in the Morris water maze via attenuating cortical oxidative damage in diabetic rats. Neurosci Lett 2017; 642:1-6.
48. Heidari S, Mehri S, Hosseinzadeh H. Memory enhancement and protective effects of crocin against D-galactose aging model in the hippocampus of Wistar rats. Iran J Basic Med Sci 2017; 20:1250-1259.
49. Rashedinia M, Lari P, Abnous K, Hosseinzadeh H. Protective effect of crocin on acrolein-induced tau phosphorylation in the rat brain. Acta Neurobiol Exp 2015; 75:208-219.
50. Tamaddonfard E, Farshid AA, Asri-Rezaee S, Javadi S, Khosravi V, Rahman B, et al. Crocin improved learning and memory impairments in streptozotocin-induced diabetic rats. Iran J Basic Med Sci 2013; 16:91-100.
51. Conrad CD. What is the functional significance of chronic stress-induced CA3 dendritic retraction within the hippocampus? Behav Cogn Neurosci Rev 2006; 5:41-60.
52. Conrad CD. A critical review of chronic stress effects on spatial learning and memory. Prog Neuropsychopharmacol Biol Psychiatry 2010; 34:742-755.
53. Ghadrdoost B, Vafaei AA, Rashidy-Pour A, Hajisoltani R, Bandegi AR, Motamedi F, et al. Protective effects of saffron extract and its active constituent crocin against oxidative stress and spatial learning and memory deficits induced by chronic stress in rats. Eur J Pharmacol 2011; 667:222-229.
54. Hosseinzadeh H, Sadeghnia HR, Ghaeni FA, Motamedshariaty VS, Mohajeri SA. Effects of saffron (Crocus sativus L.) and its active constituent, crocin, on recognition and spatial memory after chronic cerebral hypoperfusion in rats. Phytother Res 2012; 26:381-386.
55. Hadizadeh F, Mohajeri S, Seifi M. Extraction and purification of crocin from saffron stigmas employing a simple and efficient crystallization method. Pak J Biol Sci 2010; 13: 691-698.
56. Bejar C, Wang R-H, Weinstock M. Effect of rivastigmine on scopolamine-induced memory impairment in rats. Eur J Pharmacol 1999; 383:231-240.
57. Razavi BM, Hosseinzadeh H, Movassaghi AR, Imenshahidi M, Abnous K. Protective effect of crocin on diazinon induced cardiotoxicity in rats in subchronic exposure. Chem Biol Interact 2013; 203:547-555.
58. Jiang X, Chai G-S, Wang Z-H, Hu Y, Li X-G, Ma Z-W, et al. CaMKII-dependent dendrite ramification and spine generation promote spatial training-induced memory improvement in a rat model of sporadic Alzheimer’s disease. Neurobiol Aging 2015; 36:867-876.
59. Jamshidzadeh A, Aram M. The effects of grape seed and grape pomace extracts on spatial memory impairment induced by hyoscine in mice. J Med Plants Res 2010; 4:2334-2339.
60. Aigner TG, Mishkin M. The effects of physostigmine and scopolamine on recognition memory in monkeys. Behav Neural Biol 1986; 45:81-87.
61. Gomar A, Hosseini A, Mirazi N, Gomar M. Effect of Zingiber
Officinale (ginger rhizomes) hydroethanolic extract on
hyoscine-induced memory impairment in adult male rats.
ICNSJ 2015; 2:105-110.
62. Morris R, Anderson E, Lynch Ga, Baudry M. Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 1986; 319:774-776.
63.Tsien JZ, Huerta PT, Tonegawa S. The essential role of hippocampal CA1 NMDA receptor–dependent synaptic plasticity in spatial memory. Cell 1996; 87:1327-1338.
64. Sanderson DJ, Good MA, Seeburg PH, Sprengel R, Rawlins JNP, Bannerman DM. The role of the GluR-A (GluR1) AMPA receptor subunit in learning and memory. Prog Brain Res 2008; 169:159-178.
65. Zhang L, Fang Y, Xu Y, Lian Y, Xie N, Wu T, et al. Curcumin improves amyloid β-peptide (1-42) induced spatial memory deficits through BDNF-ERK signaling pathway. PLoS One 2015; 10: 131-525.
66.Huo X-l, Min J-j, Pan C-y, Zhao C-c, Pan L-l, Gui F-f, et al. Efficacy of lovastatin on learning and memory deficits caused by chronic intermittent hypoxia-hypercapnia: through regulation of NR2B-containing NMDA receptor-ERK pathway. PLoS One 2014; 9: 94-278.