**3. Discussion**

AD is characterized by deterioration in memory, thinking, and the ability to carry out activities. An estimated 50 million people worldwide manifest dementia, and almost 10 million new cases are reported every year [24]. There is no established therapeutic agent currently available to treat dementia. Numerous new treatments are being investigated, in various stages of clinical trials. This study suggested that injection of TMT caused critical deficits in performance during tests of cognitive function, as well as causing corresponding signs of neurodegeneration, including decreased cholinergic neurons in the hippocampus. Our results showed that administration of Bean-PS reduced the TMT-induced learning and memory deficits in the Morris water maze, and suppressed TMT-induced reduction in ChAT and AchE in the hippocampus.

TMT exposure triggers severe behavioral and cognitive defects in both humans [25] and experimental animals [26]. TMT injection induced damage to the hippocampal pyramidal neurons, and also in the associated areas in rats [26–33]. Furthermore, previous studies have reported that TMT injection increases the risk of neuronal cell death, via possible excitotoxicity, intracellular calcium overload, and mitochondrial damage [34]. Several behavioral studies have suggested that the performance of TMT-induced rats is poor in memory and learning tasks [35,36]. The Morris water maze test has been used to test permanent spatial learning ability and reference memory, and utilized to evaluate cognitive-enhancing agents for the treatment of neurocognitive disorders [37,38].

Our study indicated that spatial memory impairment was ameliorated in the TMT+Bean-PS groups during the training days in contrast with that of the TMT group. In addition, the data demonstrated that Bean-PS preserved the TMT-induced reduction in spatial retention. These results suggested that Bean-PS improved learning and memory deficits in TMT-intoxicated rats used as experimental models of neurodegeneration for the study of Alzheimer-like diseases [15,39].

The animal model used in this study clearly demonstrates the functional significance of hippocampal neurodegeneration induced by TMT. The cholinergic system is involved in information processing associated with hippocampal learning and memory [40]. The hippocampus carries information derived from the related regions of the brain that are involved in learning and memory [41], and any damage to the cholinergic system may result in altered behavioral responses [42]. In particular, the loss of cholinergic function has been associated with a decline in cognition during aging and in AD [43]. Thus, the expression of AchE and ChAT in the hippocampus and its relation to TMT-induced cognitive impairment in rats was examined. Bean-PS also continuously improved the activity of cholinergic neurons in the hippocampus, which eventually restored the cholinergic pathway [44].

Based on the cholinergic hypothesis, patients with senile dementia show a selective and irreversible lack of cholinergic function in the brain [45]. Therefore, in patients with AD, treatment with cholinesterase inhibitors and ChAT activators may compensate for decreased ACh levels.

It is quite probable that the observed improvement in spatial learning deficits following the treatment with Bean-PS of rats was related to the enhanced release of ACh. According to the results from the Morris water maze (MWM), exposure to Bean-PS ameliorated the TMT-induced deficits in learning and memory. In addition, treatment with Bean-PS decreased cell loss, increased central cholinergic function, and prevented degeneration of cholinergic neurons mediating cognitive processes [46].

Our previous study reported that treatment with Bean-PS dissolved in medium-chain triglyceride (MCT) improved cognitive function and enhanced the neural activity in rats with TMT-induced learning and memory deficits. The active biological ingredient in the resulting extract is affected by the solvents [47]. Therefore, we investigated how the activity of Bean-PS in 10% hexane solvent affected TMT-induced memory deficits in rats.

The present study analyzed the effect of 10% hexane solvent on Bean-PS-attenuated TMT-induced cognitive defects in the Morris water maze, and found a protective effect in contrast to TMT-induced reduction in ChAT and AchE in the hippocampal areas.

Exposure to Bean-PS resulted in upregulation of glucose uptake in the hippocampus and frontal lobe. Administration of Bean-PS may have robust therapeutic potential as a treatment for neurodegenerative disorders, such as AD.

## **4. Material and Methods**
