We hypothesized whether CAE and nobiletin treatments have similar anti-dementia effects and underlying anti-apoptotic activity against β-amyloid-induced memory impairments in mice.

The CAE was supplied by KPLC Group Paris, France and prepared as described previously. The main component of CAE, nobiletin, was analyzed by high-performance liquid chromatography according to followed method: The solvent was a mixture of water A and Methanol B and delivered at 0.

Osaka, Japan. Seongnam, South Korea. After a 7-day acclimation period, they were used in the experiment. All experiments were approved by an Ethics Committee no. Yongin, Korea and performed in accordance with the national guidelines for the care and use of laboratory animals and the mice were maintained according to their guidelines.

We monitored changes in body weight once a week, and observed changes in feed and water intake. In additions, appearance changes such as hair coat, activity, and posture were monitored once a day after β-amyloid injection over the course of the experiments.

To improve animal well-being, we provided a sanitary environment to prevent disease and proper breeding and management and used appropriate painkillers and anesthetics to reduce pain. The humane endpoints were set by observing body weight change, hair coat, movement and posture of mice and applied in consultation with experimental committee.

When both sings as lack of respiration and faded eye color were observed, the mice were removed from the CO 2 chamber.

Aβ 1—42 was dissolved in sterile 0. The respective drugs were given orally p. for 4 weeks Day Based on the results of previous studies, the concentrations of CAE and nobiletin were set 3 , 13 , The passive avoidance test was conducted in all groups for 3 days Days 15—17 and Morris water maze MWM task for 7 days Days 22— The mice were performed by cardiac puncture for exsanguination after carbon dioxide anesthesia at Day 28 and were confirmed the death by observing stop breathing and cardiac dysfunction through CO 2 anesthesia and exsanguination.

The entire experimental schedule is shown in Fig. To determine learning and memory ability, the passive avoidance test was conducted in an acrylic shuttle box with two compartments and a guillotine door in the middle.

The box consists of illuminated and non-illuminated compartments with an electric grid floor that allows for electronic shock stimuli. The test was performed on 3 consecutive days at h intervals.

On the first day Day 15 , the animals were allowed to explore the non-illuminated compartment for 2 min and then transferred back to the illuminated compartment. They were then allowed to access the illuminated and non-illuminated compartment freely for 60 sec.

If the mice moved to the non-illuminated compartment, they were immediately removed and trained to adapt to the illuminated compartment. After the end of adaptation the time, on the second day, the mice moved between the two compartments for sec, but when they entered the non-illuminated compartment, the guillotine door automatically closed and a scrambled shock was given for 2 sec.

On the last day of the test, Day 17, the mice were placed in the illuminated compartment and the time taken for them to move to the non-illuminated compartment after the door opened was measured.

On Day 22, the mice were tested using the MWM task. A circular water pool was divided into quadrants; the platform was randomly located within one quadrant. The mice were trained to find the platform for 60 sec. When the mice reached the platform, they were allowed to remain on the platform for 30 sec; if they could not find the platform within 60 sec, they were guided and placed on it for 30 sec to learn the extra maze cues.

All animals performed two trials per day and the position of the platform in the circular pool was randomly changed. The task consisted of 2 days of training; 4 days of acquisition, during which the time to find the platform was recorded; and 1 day of probe trial. For the probe trial, the platform was removed and the animals were allowed to search it for 1 min.

The number of crossings by the mice in the quadrant with the removed platform was measured on Day At the end of the experiment, the mice were sacrificed, and the cortex and hippocampus were dissected from the brain.

Both tissues were rapidly homogenized and sonicated in 0. AchE activity was determined using commercial assay kits Abnova; cat. KA, Taiwan according to the manufacturer's instructions.

The activity was calculated as the optical density OD at nm and represented as OD values per milligram of protein. The dissected cortex and hippocampus tissues were lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris-HCl pH 7. The supernatant was collected, and the protein concentration was calculated by bicinchoninic acid protein assay kit Thermo, USA.

The membranes were blocked with commercial blocking buffer Thermo, USA for 1 h at room temperature and washed thrice with Tris-buffered saline containing 0. After washing, the membrane was incubated at 4°C overnight with the following appropriate antibodies: B-cell lymphoma 2 Bcl-2 -associated X protein Bax; ,; cat.

The membranes were again washed thrice and enhanced using chemi-luminescence reagents. The protein bands on the membrane were detected by a chemi-luminometer ATTO, Japan. Densitometry was performed using the Image-Pro Plus soft-ware version 6.

Data are expressed as the mean ± standard error and were analyzed with SPSS Statistics Different treatment groups were compared using one-way analysis of variance followed by multiple comparisons using Dunnett's post hoc test using Origin 7. The composition in CAE were investigated the chromatographic profiles of a standard on HPLC analysis.

The optimized CAE was used in all subsequent experiments. The mice did not show any change in body weight during the experiment, but hair coat showed rough condition. Response to weak stimuli decreased and activity ability also decreased.

Also, the mice was sitting on the floor with a curved posture was observed and through these symptoms, the humane endpoint was set. The animals were sacrificed by meeting the defined endpoint. The effect of CAE and nobiletin on the Aβ 1—induced memory impairment was measured using a passive avoidance task.

The step-through latency of the Aβ 1—only treated group was significantly shortened compared to the control group Fig. However, the reduced step latency with Aβ 1—42 was restored by the administration of CAE and nobiletin. Effect of nobiletin and CAE on step-through latency in passive avoidance test in Aβ 1—42 induced memory impairment.

The data are presented as mean ± standard error of the mean. Aβ, amyloid β; CAE, Citrus aurantium extract. The efficacy of CAE and nobiletin in protection from the spatial memory impairment via Aβ 1—42 injection was further confirmed.

The escape latency assessment was performed twice a day. During the test period, escape latency decreased slightly in the second trial compare to the first trial in all experimental groups Fig.

No difference was observed in the escape latency for 4 days in the amnesic mice, which were treated with Aβ 1— By contrast, the control group showed significantly decreased escape latency in two trials over 4 days Fig. It is well-established that Aβ 1—42 induces memory loss and increases the escape latency.

Similarly, the escape latency in mice administered nobiletin significantly decreased for 4 days compared to the Aβ 1—only injected group. Effect of nobiletin and CAE on Aβ 1—42 induced memory impairment in the Morris water maze. The escape latency of A Trial 1 and B Trial 2, and the mean of C Trial 1 and Trial 2 during the training sessions for 4 days.

The effect of CAE and nobiletin treatment on the swim distance to locate the platform in the MWM task is shown in Table I. The control group mice were able to swiftly locate the platform and reached the platform during the training session.

However, the Aβ 1—treated group had difficulty learning to locate the platform. The swim distance was significantly increased compared to that of the control group. The mice in the nobiletin-treated group were also able to find the platform easily with a short swim distance, especially on Day Effect of nobiletin and CAE on the distance swum by Aβ 1—42 treated mice to find the platform in the water maze task.

To evaluate the spatial memory of the mice, the number of crossings to the platform was measured in the probe trial on Day As shown in Fig. These results show that these drugs enhance spatial cognition, learning, and memory functions against Aβ 1—induced memory impairment.

Effect of nobiletin and CAE on the number of crossing in a probe trail on Day To investigate the neuroprotective effect of CAE and nobiletin on brain tissue, AchE activity was measured in the cortex and hippocampus Table II. The AchE activity in the Aβ 1—treatment group was significantly increased compared to that in control group.

Similarly, the AchE activity in the nobiletin treatment group also decreased significantly by The effect of CAE and nobiletin on the Bcl-2 family and caspase pathway was investigated in the cortex and hippocampus.

In contrast, Bcl-2 protein expression was higher in the control group than in the Aβ 1—only treated group in the cortex and hippocampus. A similar protein expression pattern was observed in the cortex. Effect of nobiletin and CAE on protein expression in A hippocampal and B cortex tissues.

The expression was detected by western blot analysis. Bax, Bcl-2 and cleaved caspase-3 protein levels were normalized by separate control β-actin, respectively. Aβ, amyloid β; CAE, Citrus aurantium extract; Nob, Nobiletin. The present study is the first report to evaluate the neuroprotective effects in Aβ 1—induced memory impairment animal model and not the transgenic or senescence accelerated mouse model.

Our results showed that the Aβ 1—injection resulted in severe performance deficits in the passive avoidance and Morris water task as well as neurodegeneration in the mice brain that was evident from increased AchE activity in the hippocampus and cortex.

In this study, we treated the amnesic mice with CAE and nobiletin and confirmed the anti-amnesic effect by regulating of apoptotic signaling.

Aβ plays a major role in the development of AD, particularly the neurotoxic Aβ 1—42 The direct injection of Aβ 1—42 in the rodent brain has been used to cause apparent memory deficits, and Aβ-exposed rats have shown hippocampus-dependent spatial learning dysfunction in long and short-term tasks 4.

Also, the brains of AD patients demonstrated a high Aβ level compared with normal aged brain samples The deposition of Aβ in the cortex and hippocampus, which are responsible for learning and memory performance, resulted in neuronal apoptosis 21 , To examine the protective effect of CAE and nobiletin, we performed the passive avoidance and MWM tasks to investigate learning and memory function.

The passive avoidance task is a method that is used to measure the escape time from the space that induces pain and fear by electronic shock in rodents It is commonly used to confirm the memory function, and we found in this study that CAE and nobiletin administration significantly increased the step-through latency to similar levels, a phenomenon that was reduced by the Aβ 1—42 injection.

The MWM is an assessment method to evaluate hippocampal-dependent learning abilities and cognitive deficits in rodents. The animals were trained to learn spatial working information at the learning stage and assisted to build future memory These results are consistent with those of previous studies that show Aβ-induced memory deficits in an MWM task than those in the saline group As a result of two trials for 4 days on the MWM task, CAE treatment reduced escape latency in the second trial compared to the first trial, and escape time decreased over training days.

The nobiletin administration group showed similar escape latency for 4 days in the first trial, and the escape latency decreased rapidly on Day 26 in the second trial. Although the pattern of escape latency of the CAE and nobiletin group was slightly different, the mean escape latency was decreased to a similar pattern.

This means that CAE and nobiletin administration showed significant decreases in escape latency, improvement in cognitive performance, and amelioration of the memory deficits. Furthermore, to investigate the neuroprotective effect of CAE and nobiletin, we examined the changes in the Ach system in the hippocampus and cortex.

Ach is an essential enzyme that maintains the normal function of the nervous system and is hydrolyzed by AchE.

In addition, Aβ deposition is increased in the presence of AchE, and AchE activity in AD is related to Aβ deposition Therefore, it is important to reduce the level of AchE, which is used as a marker for the cholinergic nervous system.

Here we found that AchE activity in the cortex was similar to that of CAE and nobiletin. However, nobiletin administration showed significantly lower AchE activity than CAE administration in the hippocampus. CAE and nobiletin administration benefits on the cholinergic neurotransmission by decreasing AchE activities in the cortex and hippocampus.

AchE can also be used as a marker of apoptosis. AchE expression or activity is increased when the cells undergo apoptosis, and enhanced AchE expression levels are detected in the brain of focal cerebral ischemic rats 26 , AchE is usually present in the cytoplasm and moves to the nucleus before nuclear morphological changes occur.

It then accelerates chromatin condensation and fragmentation by modulating nuclear components Therefore, AchE can be detected on the fragmented nuclei of apoptotic cells, and increased AchE activity implies the occurrence of cell death Apoptosis is triggered via two major pathways: The mitochondrial intrinsic pathway and the death receptor-mediated extrinsic pathway.

In this study, we focused on the mitochondrial pathway, which is regulated by Bcl-2 family and caspases Bcl-2 is a known anti-apoptotic protein, while Bax is a pro-apoptotic protein that promotes apoptosis.

aurantium L. bergamia Risso and Poit. Peel essential oils. J Food Sci 77 1 — Jazayeri B, Amanlou A, Ghanadian N, Pasalar P, Amanlou M A preliminary investigation of anticholinesterase activity of some Iranian medicinal plants commonly used in traditional medicine.

DARU J PharmSci Rabiei Z, Rafieian-kopaei M, Heidarian E, Saghaei E, Mokhtari S Effects of Zizyphus jujube extract on memory and learning impairment induced by bilateral electric lesions of the nucleus basalis of meynert in rat.

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Curr Alzheimer Res 2 3 — Yamada N, Hattori A, Hayashi T, Nishikawa T, Fukuda H, Fujino T Improvement of scopolamine-induced memory impairment by Z-ajoene in the water maze in mice. Pharmacol Biochem Behav — Brain Res 1—2 — Nakajima A, Yamakuni T, Matsuzaki K, Nakata N, Onozuka H, Yokosuka A et al Nobiletin, a citrus flavonoid, reverses learning impairment associated with N-methyl-D-aspartate receptor antagonism by activation of extracellular signal-regulated kinase signaling.

J Pharm Exp Ther 2 — Carvalho-Freitas MIR, Costa M Anxiolytic and sedative effects of extracts and essential oil from Citrus aurantium L.

Biol Pharm Bull 25 12 — Vazquez J, Baghdoyan HA Muscarinic and GABA A receptors modulate acetylcholine release in feline basal forebrain.

Eur J Neurosci — Download references. This study was conducted with the assistance of Medical Plants Research Center of Shahrekord University of Medical Science and was approved by the Ethics Committee of Shahrekord University of Medical Sciences.

Authors also would like to thank Research and Technology Deputy of Shahrekord University of Medical Sciences for their financial support. Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran.

Department of Biostatistics and Epidemiology, Health Faculty, Shahrekord University of Medical Sciences, Shahrekord, Iran.

You can also search for this author in PubMed Google Scholar. Correspondence to Mahmoud Rafieian-kopaei. Reprints and permissions. Rahnama, S. et al. Anti-amnesic activity of Citrus aurantium flowers extract against scopolamine-induced memory impairments in rats.

Neurol Sci 36 , — Download citation. Received : 16 August Accepted : 24 October Published : 04 November Issue Date : April Anyone you share the following link with will be able to read this content:.

Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Access this article Log in via an institution. Alzheimers Dement 7 2 — Article CAS PubMed Google Scholar Rezvani AH, Cauley M, Sexton H, Xiao Y, Brown ML, Paige MA et al Sazetidine-A, a selective α4β2 nicotinic acetylcholine receptor ligand: effects on dizocilpine and scopolamine-induced attentional impairments in female Sprague-Dawley rats.

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J Food Sci 77 1 —46 Article Google Scholar Jazayeri B, Amanlou A, Ghanadian N, Pasalar P, Amanlou M A preliminary investigation of anticholinesterase activity of some Iranian medicinal plants commonly used in traditional medicine.

Graham T, Spriet L. Metabolic, catecholamine, and exercise performance responses to various doses of caffeine. Greer F, McLean C, Graham T. Caffeine, performance, and metabolism during repeated Wingate exercise tests. Crowe MJ, Leicht AS, Spinks WL. Physiological and cognitive responses to caffeine during repeated, high-intensity exercise.

Int J Sport Nutr Exerc Metab. Xhyheri B, Manfrini O, Mazzolini M, Pizzi C, Bugiardini R. Heart rate variability today. Prog Cardiovasc Dis. Haller CA, Duan M, Jacob P, Benowitz N. Human pharmacology of a performance-enhancing dietary supplement under resting and exercise conditions.

Br J Clin Pharmacol. Kliszczewicz BM, Esco MR, Quindry JC, Blessing DL, Oliver GD, Taylor KJ, Price BM. Autonomic responses to an acute bout of high-intensity body weight resistance exercise vs.

treadmill running. Reimann M, Rudiger H, Weiss N, Ziemssen T. Acute hyperlipidemia but not hyperhomocysteinemia impairs reflex regulation of the cardiovascular system.

Atherosclerosis Supp. Download references. The data sets used during the current study are available from the corresponding author upon reasonable request. Department of Exercise Science and Sport Management, Kennesaw State University, Kennesaw, GA, USA. You can also search for this author in PubMed Google Scholar.

BK contributed to study design, data collection HRV and Biomarker , data analysis, major contribution to the writing of the manuscript. EB contributed to data collection, performed HRV analysis and interpretation, blood assay analysis, conducted literature review, and major contribution to the writing of the manuscript.

CW contributed with data collection, assisted with data analysis Biomarker , and moderate contributions to the editing of the manuscript. PB contributed to study design, data collection, moderate editing of the manuscript. WH significant contribution to data collection, moderate editing of the manuscript.

JM contributed to study design, data statistical analysis, and moderate editing of manuscript. CM contributed to the study design, data collection, moderate editing of manuscript, and procurement of funds.

All authors read and approved the final manuscript. Correspondence to Brian Kliszczewicz. The Institutional Review Board approved all testing procedures and protocols prior to beginning data collection 17— Participants read and sign an informed consent prior too participating in this study.

These authors declare that they have no competing interest and have no relation too the supplement or associated companies. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open Access This article is distributed under the terms of the Creative Commons Attribution 4. Reprints and permissions. Kliszczewicz, B. et al. The influence of citrus aurantium and caffeine complex versus placebo on the cardiac autonomic response: a double blind crossover design.

J Int Soc Sports Nutr 15 , 34 Download citation. Received : 01 February Accepted : 16 July Published : 24 July Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative. Skip to main content. Search all BMC articles Search. Download PDF. Download ePub. Research article Open access Published: 24 July The influence of citrus aurantium and caffeine complex versus placebo on the cardiac autonomic response: a double blind crossover design Brian Kliszczewicz ORCID: orcid.

Methods Ten physically active males Background The cultivation of commercially available supplements has substantially increased throughout recent years, making the use of pharmacologic ergogenic aids more prevalent and readily available to the general population and athletic community.

Methods Participants Fourteen apparently healthy males who habitually consume caffeine 95— mg serving per day, at least 4 days a week were recruited for this study.

Experimental design The study was performed in a double-blind, placebo-controlled, randomized crossover fashion in which only one investigator knew the contents of the supplementation; this investigator was not involved in the collection or analysis of the study outcome measures. Study Design.

Full size image. Results Four participants were removed from the study due to adverse reactions to the phlebotomy procedure i. Table 2 Markers of ANS activity during the Ingestion A and Recovery Periods B Full size table.

Recovery period Following the exhaustive protocols, HR was significantly elevated in both trials and recovered in a similar time-dependent fashion Table 2A and B.

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Availability of data and materials The data sets used during the current study are available from the corresponding author upon reasonable request. Author information Author notes Brian Kliszczewicz and Emily Bechke contributed equally to this work. View author publications.

Ethics declarations Ethics approval and consent to participate The Institutional Review Board approved all testing procedures and protocols prior to beginning data collection 17— Participants read and sign an informed consent prior too participating in this study.

Consent for publication Not Applicable. Competing interests These authors declare that they have no competing interest and have no relation too the supplement or associated companies.

Rights and permissions Open Access This article is distributed under the terms of the Creative Commons Attribution 4. About this article. Cite this article Kliszczewicz, B. Ballard CG, Greig NH, Guillozet-Bongaarts AL, Enz A, Darvesh S Cholinesterases: roles in the brain during health and disease.

Curr Alzheimer Res 2 3 — Yamada N, Hattori A, Hayashi T, Nishikawa T, Fukuda H, Fujino T Improvement of scopolamine-induced memory impairment by Z-ajoene in the water maze in mice.

Pharmacol Biochem Behav — Brain Res 1—2 — Nakajima A, Yamakuni T, Matsuzaki K, Nakata N, Onozuka H, Yokosuka A et al Nobiletin, a citrus flavonoid, reverses learning impairment associated with N-methyl-D-aspartate receptor antagonism by activation of extracellular signal-regulated kinase signaling.

J Pharm Exp Ther 2 — Carvalho-Freitas MIR, Costa M Anxiolytic and sedative effects of extracts and essential oil from Citrus aurantium L. Biol Pharm Bull 25 12 — Vazquez J, Baghdoyan HA Muscarinic and GABA A receptors modulate acetylcholine release in feline basal forebrain. Eur J Neurosci — Download references.

This study was conducted with the assistance of Medical Plants Research Center of Shahrekord University of Medical Science and was approved by the Ethics Committee of Shahrekord University of Medical Sciences. Authors also would like to thank Research and Technology Deputy of Shahrekord University of Medical Sciences for their financial support.

Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran. Department of Biostatistics and Epidemiology, Health Faculty, Shahrekord University of Medical Sciences, Shahrekord, Iran.

You can also search for this author in PubMed Google Scholar. Correspondence to Mahmoud Rafieian-kopaei. Reprints and permissions.

Rahnama, S. et al. Anti-amnesic activity of Citrus aurantium flowers extract against scopolamine-induced memory impairments in rats. Neurol Sci 36 , — Download citation.

Received : 16 August Accepted : 24 October Published : 04 November Issue Date : April Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative.

Access this article Log in via an institution. Alzheimers Dement 7 2 — Article CAS PubMed Google Scholar Rezvani AH, Cauley M, Sexton H, Xiao Y, Brown ML, Paige MA et al Sazetidine-A, a selective α4β2 nicotinic acetylcholine receptor ligand: effects on dizocilpine and scopolamine-induced attentional impairments in female Sprague-Dawley rats.

Psychopharmacology 4 — Article CAS PubMed Google Scholar Hashimoto M, Hashimoto T, Kuriyama K Protective effect of WEB FU on AF64A ethylcholine aziridinium ion -induced impairment of hippocampal cholinergic neurons and learning acquisition.

Eur J Pharmacol 1 :9—14 Article CAS PubMed Google Scholar Kvaltinova Z, Juranek I, Machova J, Stolc S Effect of oxidative stress on 3H N-methylscopolamine binding and production of thiobarbituric acid reactive substances in rat cerebral cortex membranes.

Gen Physiol Biophys — CAS PubMed Google Scholar Vasco VRL Phosphoinositide pathway and the signal transduction network in neural development.

The humane endpoints were set by observing body weight change, hair coat, movement and posture of mice and applied in consultation with experimental committee.

When both sings as lack of respiration and faded eye color were observed, the mice were removed from the CO 2 chamber. Aβ 1—42 was dissolved in sterile 0. The respective drugs were given orally p. for 4 weeks Day Based on the results of previous studies, the concentrations of CAE and nobiletin were set 3 , 13 , The passive avoidance test was conducted in all groups for 3 days Days 15—17 and Morris water maze MWM task for 7 days Days 22— The mice were performed by cardiac puncture for exsanguination after carbon dioxide anesthesia at Day 28 and were confirmed the death by observing stop breathing and cardiac dysfunction through CO 2 anesthesia and exsanguination.

The entire experimental schedule is shown in Fig. To determine learning and memory ability, the passive avoidance test was conducted in an acrylic shuttle box with two compartments and a guillotine door in the middle. The box consists of illuminated and non-illuminated compartments with an electric grid floor that allows for electronic shock stimuli.

The test was performed on 3 consecutive days at h intervals. On the first day Day 15 , the animals were allowed to explore the non-illuminated compartment for 2 min and then transferred back to the illuminated compartment.

They were then allowed to access the illuminated and non-illuminated compartment freely for 60 sec. If the mice moved to the non-illuminated compartment, they were immediately removed and trained to adapt to the illuminated compartment.

After the end of adaptation the time, on the second day, the mice moved between the two compartments for sec, but when they entered the non-illuminated compartment, the guillotine door automatically closed and a scrambled shock was given for 2 sec. On the last day of the test, Day 17, the mice were placed in the illuminated compartment and the time taken for them to move to the non-illuminated compartment after the door opened was measured.

On Day 22, the mice were tested using the MWM task. A circular water pool was divided into quadrants; the platform was randomly located within one quadrant. The mice were trained to find the platform for 60 sec. When the mice reached the platform, they were allowed to remain on the platform for 30 sec; if they could not find the platform within 60 sec, they were guided and placed on it for 30 sec to learn the extra maze cues.

All animals performed two trials per day and the position of the platform in the circular pool was randomly changed. The task consisted of 2 days of training; 4 days of acquisition, during which the time to find the platform was recorded; and 1 day of probe trial.

For the probe trial, the platform was removed and the animals were allowed to search it for 1 min. The number of crossings by the mice in the quadrant with the removed platform was measured on Day At the end of the experiment, the mice were sacrificed, and the cortex and hippocampus were dissected from the brain.

Both tissues were rapidly homogenized and sonicated in 0. AchE activity was determined using commercial assay kits Abnova; cat. KA, Taiwan according to the manufacturer's instructions. The activity was calculated as the optical density OD at nm and represented as OD values per milligram of protein.

The dissected cortex and hippocampus tissues were lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris-HCl pH 7.

The supernatant was collected, and the protein concentration was calculated by bicinchoninic acid protein assay kit Thermo, USA. The membranes were blocked with commercial blocking buffer Thermo, USA for 1 h at room temperature and washed thrice with Tris-buffered saline containing 0.

After washing, the membrane was incubated at 4°C overnight with the following appropriate antibodies: B-cell lymphoma 2 Bcl-2 -associated X protein Bax; ,; cat. The membranes were again washed thrice and enhanced using chemi-luminescence reagents. The protein bands on the membrane were detected by a chemi-luminometer ATTO, Japan.

Densitometry was performed using the Image-Pro Plus soft-ware version 6. Data are expressed as the mean ± standard error and were analyzed with SPSS Statistics Different treatment groups were compared using one-way analysis of variance followed by multiple comparisons using Dunnett's post hoc test using Origin 7.

The composition in CAE were investigated the chromatographic profiles of a standard on HPLC analysis. The optimized CAE was used in all subsequent experiments.

The mice did not show any change in body weight during the experiment, but hair coat showed rough condition. Response to weak stimuli decreased and activity ability also decreased. Also, the mice was sitting on the floor with a curved posture was observed and through these symptoms, the humane endpoint was set.

The animals were sacrificed by meeting the defined endpoint. The effect of CAE and nobiletin on the Aβ 1—induced memory impairment was measured using a passive avoidance task.

The step-through latency of the Aβ 1—only treated group was significantly shortened compared to the control group Fig. However, the reduced step latency with Aβ 1—42 was restored by the administration of CAE and nobiletin. Effect of nobiletin and CAE on step-through latency in passive avoidance test in Aβ 1—42 induced memory impairment.

The data are presented as mean ± standard error of the mean. Aβ, amyloid β; CAE, Citrus aurantium extract. The efficacy of CAE and nobiletin in protection from the spatial memory impairment via Aβ 1—42 injection was further confirmed. The escape latency assessment was performed twice a day.

During the test period, escape latency decreased slightly in the second trial compare to the first trial in all experimental groups Fig. No difference was observed in the escape latency for 4 days in the amnesic mice, which were treated with Aβ 1— By contrast, the control group showed significantly decreased escape latency in two trials over 4 days Fig.

It is well-established that Aβ 1—42 induces memory loss and increases the escape latency. Similarly, the escape latency in mice administered nobiletin significantly decreased for 4 days compared to the Aβ 1—only injected group.

Effect of nobiletin and CAE on Aβ 1—42 induced memory impairment in the Morris water maze. The escape latency of A Trial 1 and B Trial 2, and the mean of C Trial 1 and Trial 2 during the training sessions for 4 days. The effect of CAE and nobiletin treatment on the swim distance to locate the platform in the MWM task is shown in Table I.

The control group mice were able to swiftly locate the platform and reached the platform during the training session. However, the Aβ 1—treated group had difficulty learning to locate the platform. The swim distance was significantly increased compared to that of the control group. The mice in the nobiletin-treated group were also able to find the platform easily with a short swim distance, especially on Day Effect of nobiletin and CAE on the distance swum by Aβ 1—42 treated mice to find the platform in the water maze task.

To evaluate the spatial memory of the mice, the number of crossings to the platform was measured in the probe trial on Day As shown in Fig. These results show that these drugs enhance spatial cognition, learning, and memory functions against Aβ 1—induced memory impairment.

Effect of nobiletin and CAE on the number of crossing in a probe trail on Day To investigate the neuroprotective effect of CAE and nobiletin on brain tissue, AchE activity was measured in the cortex and hippocampus Table II. The AchE activity in the Aβ 1—treatment group was significantly increased compared to that in control group.

Similarly, the AchE activity in the nobiletin treatment group also decreased significantly by The effect of CAE and nobiletin on the Bcl-2 family and caspase pathway was investigated in the cortex and hippocampus.

In contrast, Bcl-2 protein expression was higher in the control group than in the Aβ 1—only treated group in the cortex and hippocampus. A similar protein expression pattern was observed in the cortex.

Effect of nobiletin and CAE on protein expression in A hippocampal and B cortex tissues. The expression was detected by western blot analysis. Bax, Bcl-2 and cleaved caspase-3 protein levels were normalized by separate control β-actin, respectively.

Aβ, amyloid β; CAE, Citrus aurantium extract; Nob, Nobiletin. The present study is the first report to evaluate the neuroprotective effects in Aβ 1—induced memory impairment animal model and not the transgenic or senescence accelerated mouse model.