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Objectives: To study effects of willughbela cochinchinensis (WC) on learning and memory deficits in experimental animals. Subjects and methods: 50 Swiss mice were randomly separated into 5 experimental groups, 10 mice for each group.
Journal of military pharmaco-medicine No7-2017 AMELIORATION OF LEARNING AND MEMORY DEFICITS BY WILLUGHBEIA COCHINCHINENSIS IN MICE Nguyen Thi Hoa*; Le Van Quan*; Can Van Mao* SUMMARY Objectives: To study effects of willughbela cochinchinensis (WC) on learning and memory deficits in experimental animals Subjects and methods: 50 Swiss mice were randomly separated into experimental groups, 10 mice for each group Group 1: mice were intraperitoneally (i.p) injected and orally (p.o) administered saline at dose 0.1 mL/10 g; group 2: mice were injected i.p 1.5 mg/kg scopolamin and p.o 0.1 mL/10 g saline; group 3, group and group 5: mice were injected i.p 1.5 mg/kg scopolamin and p.o 100 mg/kg, 150 mg/kg and 200 mg/kg WC, respectively 60 minutes after drug injections, animals performed a passive avoidance test which includes two phases: training phase: animals were placed in the light compartment and if they moved to the dark compartment, they were given electrical foot shocks for seconds; test phase: animals were also placed in the light compartment but they were not given any electrical foot shocks at the dark compartment Results: In test phase, mean latency to entry dark compartment in group was shorter than that in group and the latencies in group and were longer than that in group Conclusion: Our results provided an evidence for effective treatment of WC in memory deficits animal model * Keyword: Willughbela cochinchinensis; Learning and memory deficits; Mice INTRODUCTION Alzheimer’s disease (AD) accounts for 60 - 80% of cases of dementia in older people [1] Mechanism of AD has been suggested to be involved in neurodegeneration and formations of plaques and neurofibrillary tangles in brains which cause atrophied cortex and enlarged ventricles [2] Following these damages in brains, patients with AD develop deficits in memory, recognition and behavioral controlling [3] If they don’t receive any treatments, these disorders will be worse and seriously affect to their life as well as their families Up to date, there is no effective special treatments for AD and patients with AD must receive treatments in all their life Furthermore, in some cases, effects of drugs on AD treatments are limited Thus, development of new drugs and natural plants for effective treatments of AD are necessary It has indicated that dementia in patients with AD relates to disorders in cholinergic systems Thus, they used scopolamin, cholinergic receptor antagonist, to induce a animal model of AD WC or “Gui do” has been used in Vietnamese traditional medicines used for treatment of dementia * Vietnam Military Medical University Corresponding author: Can Van Mao (canvanmao2011@gmail.com) Date received: 10/06/2017 Date accepted: 10/08/2017 42 Journal of military pharmaco-medicine no7-2017 as well as diarrhea, heartburn, and subcutaneous abscess and as a diuretic Our preliminary screening study revealed that methanolic extracts derived from the wood of W cochinchinensis exhibit AChE (acetylcholinesterase) and BChE (butyrylcholinesterase) strong inhibited activities which are main mechanism of actions of drugs for treatments of AD [4] To provide basics for using WC to treat AD in humans, we conducted the present study with the aims: To investigate effects of WC on deficits in learning and memory in experimental animals SUBJECTS AND METHODS Subjects 50 Swiss mice (150 - 250 g body weight) were used in the present study Animals were housed in individual cages, maintained in controlled temperature and 12h light/dark cycles with free access to water and food The present study was conducted at Department of Physiology, Vietnam Military Medical University All procedures were performed in accordance with the Animal Center Guidelines for the Care and Use of Laboratory Animals at the Vietnam Military Medical University * Materials: WC was isolated by Department of Pharmacy, Hochiminh City University of Medicine and Pharmacy and was supplied in power form WC power was dissolved in saline using a magnetic stirrer Methods * Animal grouping and drug treatments: Animals were separated randomly into experimental groups, 10 mice for each group Group (control group): mice were ip and p.o treated saline; group (scopolamin group): mice were i.p treated scopolamin 1.5 mg/kg and p.o treated saline at 0.1 mL/10 g; group 3, group and group (WC groups): mice were i.p injected scopolamin 1.5 mg/kg and p.o WC 100 mg/kg, 150 mg/kg and 200 mg/kg, respectively WC and saline were orally administered at 60 minutes and scopolamin and saline were i.p injected at 30 minutes before the behavioral task * Passive avoidance test: Animals were required to perform passive avoidance test, which includes two phases: - Training phase: was conducted at 60 minute after WC treatments on the first day Passive avoidance box (Ugo Basile) was a chamber which contained compartments: light one and dark one There was a wall with a door to separate these compartments (fig.1) The mice were placed in light compartment and explore freely for 30 minutes Then, the door was raised to allow the mice to enter the dark compartment When the mice entered the dark compartment, the door was closed and an electronic foot shock was delivered for seconds If mice didn’t entered the dark compartment within 300 seconds, mice were captured and placed inside the dark compartment and a foot shock was delivered for seconds 43 Journal of military pharmaco-medicine No7-2017 Figure 1: Apparatus of passive avoidance test - Test phase: were conducted on the second day Mice were placed in the light compartment and the door was raised In the test phase, when mice entered the dark compartment, no foot shock was delivered Mice’s behaviors were recorded for 300 seconds by using a digital video system If animals didn’t enter the dark compartment, entry latencies were measured as 300 seconds and the trials were over * Research indicators: In the present study, we analyzed some research indicators, follow as: - Latencies from beginning of trials to entrance into the dark compartment Units were measured as seconds (s) In the present study, entry latencies were analyzed in the training phase and the test phase - Average speeds, units were measures as meter/second (m/s) * Data analyses: Entry latencies and average speeds were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey’s post-hoc test for multiple comparisons, using SPSS 19.0 Results were considered to be statistically significant at p < 0.05 All results were expressed as mean ± SEM RESULTS Differences in entry latencies in the training phase Figure 2: Entry latencies in training phase 44 Journal of military pharmaco-medicine no7-2017 Figure showed mean entry latencies of experimental groups in the training phase One way ANOVA indicated that there were no significant differences in entry latencies between experimental groups in the training phase (p > 0.05) Differences in entry latencies in the test phase Figure 3: Entry latencies in test phase Figure showed differences in entry latencies between experimental groups in the test phase One way ANOVA indicated there was a significant main effects of experimental group [F(4.49) = 4.949; p = 0.002] Post hoc test indicated that mean entry latency in the scopolamin group was significantly shorter than this in the control group (Tukey test, p < 0.05) Contrarily, entry latencies in WC 150 mg/kg WC 200 mg/kg treated groups were significantly longer than that in the scopolamin treated group (p < 0.001) Differences in average speeds Figure 4: Average speeds in experimental groups 45 Journal of military pharmaco-medicine No7-2017 Figure showed differences in average speeds between experimental groups One way ANOVA indicated that there was a significant difference in average speeds between experimental groups [F(4.49) = 2.991, p = 0.029] Post hoc test indicated that mean average speed was significantly higher than that in the control group (Tukey test, p < 0.05) After WC treatments, mean average speeds in the WC 150 mg/kg group and WC 200 mg/kg group were significantly lower than that in the scopolamin group (p < 0.05) DISCUSSION Passive avoidance test is used widely to evaluate learning and memory in rodents [5] Thus, this behavioral test is appropriate for the aim of our study In the present study, in the training phase, mice had no experience with dangerous events (foot shocks) in the dark compartment Thus, mice had tendencies to move to the dark compartment because of their nocturnal life These reasons induced, there was no significant difference in entry latencies between experimental groups In the test phase, learning and memory abilities of animals were expressed When animals had these better abilities, they would recognize that when they moved to the dark compartment, they were received foot shocks Thus, the longer entry latencies they present, the better learning and memory abilities of animals they have In the present study, scopolamin induced deficits in learning and memory abilities of animals expressed by differences in entry latencies between the control group and scopolamin group Entry latencies of scopolamin treated mice were shorter 46 than these of saline treated mice These results are consisted with previous studies [6, 7] Interestingly, in the present study, we found that after WC treatments, there were a significant increases in entry latencies of animals treated by WC at doses 150 mg/kg and 200 mg/kg, in compared to that of animals treated by scopolamin These results indicated that WC ameliorated scopolamin-induced disorders in learning and memory in experimental animals Furthermore, in the present study, we also found that scopolamin induced hyperactivities in experimental animals These results are consistent with effects of scopolamin to inhibit acetylcholinesterase enzyme [8] and also with hyperactivities of patients with AD caused by disorders in activities of the cholinergic system [9] Interestingly, WC treatments also reduced locomotordisorders of experimental animals The present’s results provided a important base for us to conduct next steps to apply WC for treatments of diseases relating to deficits In learning and memory, such as Alzheimer’s disease CONCLUSION In the present study, we demonstrated that WC reduced disorders in learning and memory as well as locomotion in experimental animals: - In the test phase of passive avoidance test, WC at doses 150 mg/kg and 200 mg/kg increased entry latencies in animals with scopolamin-induced deficits in learning and memory - WC at the same doses decreased average speeds of animals with scopolamininduced hyperactivities Journal of military pharmaco-medicine no7-2017 ACKNOWLEDGEMENTS This work was supported by Grant 106-YS.05-2013.24 from Vietnam’s National Foundation for Science and Technology Development (NAFOSTED) REFERENCE Alzheimer's Association Alzheimer's disease facts and figures Alzheimers Dement 2014, 10 (2), e47-92 Madsen S.K, Gutman B.A, Joshi S.H, Toga A.W, Jack C.R Jr, Weiner M.W, Thompson P.M Mapping dynamic changes in ventricular volume onto baseline cortical surfaces in normal aging, MCI, and Alzheimer's disease Multimodal Brain Image Anal 2013, 8159, pp.84-94 Gacar N, Mutlu O, Utkan T, Komsuoglu C.I, Gocmez S.S, Ulak G Beneficial effects of resveratrol on scopolamine but not mecamylamine induced memory impairment in the passive avoidance and Morris water maze tests in rats Pharmacol Biochem Behav 2011, 99, pp.316-323 Rush D.K Scopolamine amnesia of passive avoidance: a deficit of information acquisition Behav Neural Biol 1988, 50 (3), pp.255-274 Tabrizian K, Yaghoobi N.S, Iranshahi M, Shahraki J, Rezaee R, Hashemzaei M Auraptene consolidates memory, reverses scopolamine-disrupted memory in passive avoidance task, and ameliorates retention deficits in mice Iran J Basic Med Sci 2015, 18 (10), pp.1014-1019 Desai A.K, Grossberg G.T Recognition and management of behavioral disturbances in dementia Prim Care Companion J Clin Psychiatry 2001, (3), pp.93-109 Shannon H.E, Peters S.C A comparison of the effects of cholinergic and dopaminergic agents on scopolamine-induced hyperactivity in mice J Pharmacol Exp Ther 1990, 255 (2), pp.549-553 Desmarais J.E, Gauthier S Alzheimer disease: clinical use of cholinergic drugs in Alzheimer disease Nat Rev Neurol 2010, (8), pp.418-420 Khachiyants N, Trinkle D, Son S.J, Kim K.Y Sundown syndrome in persons with dementia: an update Psychiatry Investig 2011, (4), pp.275-287 47 ... the better learning and memory abilities of animals they have In the present study, scopolamin induced deficits in learning and memory abilities of animals expressed by differences in entry latencies... mg/kg and 200 mg/kg increased entry latencies in animals with scopolamin-induced deficits in learning and memory - WC at the same doses decreased average speeds of animals with scopolamininduced... were i.p injected scopolamin 1.5 mg/kg and p.o WC 100 mg/kg, 150 mg/kg and 200 mg/kg, respectively WC and saline were orally administered at 60 minutes and scopolamin and saline were i.p injected