Antidepressant How to use: Mix drops with water in a diffuser and breathe deeply. Muscle Spasms How to use: To alleviate cramps, add drops to 10ml 2 teaspoons organic virgin coconut oil and massage gently on the affected area.

To treat spasmodic cough, massage the mixture onto your chest and neck. Repel Ants and Flies How to use: Mix drops of sweet orange essential oil with 1 cup of water in a spray bottle.

Shake well before use. Spray onto surfaces to clean, kill germs, and repel ants or flies. Relieve Inflammation How to use: Add drops to 10ml 2 teaspoons of organic virgin coconut oil.

Apply to effectively reduce inflammation. Reduce Pain How to use: Put 4 drops of sweet orange essential oil on a cotton pad and pin it to your collar, or mix drops with water in a diffuser and breathe deeply. Oil Pulling How to use: Mix 2 drops sweet orange essential oil with tablespoons of organic virgin coconut oil.

Swish the mixture in your mouth for 10 minutes. Oil pulling detoxifies, kills bacteria, prevents bad breath, tooth decay, gum disease and boosts the immune system. Boost Immunity Mix drops with water in a diffuser and diffuse throughout the home. The limonene in sweet orange essential oil makes it a powerful antioxidant.

Due to the tonic properties of this oil, it helps to strengthen all systems of the body. 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.

These two proteins are the major factors responsible for cell death regulation. The Bcl-2 and Bax ratio determines whether a cell undergoes or escapes apoptosis 23 , In our study, the Aβ 1—42 injection group had increased Bax and cleaved caspase-3 protein expressions compared with the control group, while Bcl-2 protein levels were increased in the control group and reduced in the Aβ 1—treated group.

CAE treatment significantly decreased the Bax and cleaved caspase-3 protein expression levels and simultaneously increased Bcl-2 protein expression in the hippocampus and cortex.

Likewise, the treatments also increased the expression ratio of Bcl-2 to Bax in the cortex and hippocampus.

On the other hand, Bcl-2 protein expression in the nobiletin administration group was increased to a level similar to that of the control group.

Bax and cleaved caspase-3 protein expressions in the cortex were significantly inhibited by nobiletin treatment, while the Bcl-2 protein level was significantly enhanced compared the control group. In conclusion, our results indicate that the administration of CAE and nobiletin had a similar neuroprotective effect against Aβ-induced cognitive impairment through reduction of AchE activity and anti-apoptotic activity and regulating the Bcl-2 family and caspase pathway in the cortex and hippocampus.

Our results provide evidence of the dietary intake of CAE or nobiletin as a valuable functional food since it has the ability to reduce cognitive impairment and memory dysfunction. However, to confirm the neuroprotective effects of CAE and nobiletin, we have to confirm the morphological change of brain tissues in further study.

In addition, the effects of CAE and nobiletin on Aβ accumulation in cortex and hippocampus should be studied. The present study was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries IPET through High Value-added Food Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs MAFRA; grant no.

HJL carried out the experiments and wrote the original manuscript. SKL analyzed the experimental data. DRL and BL performed the data processing and quality control assessment. BKC and SHY designed the study, and proofread and finalized the manuscript.

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