Neurogenesis Promoting Effect of Sugarcane (Saccharum officinarum L.) Top Extract and Its Cognitive Function Improvement

概要

1. Title of the Thesis/Dissertation :
Neurogenesis Promoting Effect of Sugarcane (Saccharum officinarum L.) Top Extract and Its Cognitive Function Improvement

2. Summary
Cognitive decline, such as memory loss and learning disabilities, is strongly associated with aging. The normal aging process is often accompanied by mitochondrial abnormalities, increased oxidative stress, and DNA damage. The accumulation of these processes triggers early symptoms of the neurodegenerative disease, such as cortical (especially hippocampal) atrophy, abnormalities in immune response and neurogenesis, and reduced neurotransmitter concentrations. Due to the rapidly aging population and increasing awareness of the risk of dementia, prevention and treatment of age-related cognitive decline has become a research priority. At present, pharmacological therapies are limited, and attention has recently turned to the role of dietary interventions and nutritional supplements.

Sugarcane (Saccharum officinarum L.) is widely cultivated in subtropical and tropical regions as a source of sugar. From an industrial point of view, the most profitable part of the plant is the stem, which contains a relatively high concentration of sugar. On the other hand, the top part of the plant (sugarcane top), which contains the leaves, is generally treated as a waste product. However, it has been reported that sugarcane tops contain more polyphenols with antioxidant properties than their stem. Further chemical analysis by HPLC revealed that 3-O-caffeoylquinic acid (3CQA), 5-O-caffeoylquinic acid (5CQA), 3-O-Feruloylquinic acid (3FQA), and isoorientin (luteolin-6-C-glucoside, ISO) are rich in sugarcane top ethanolic extract (STEE), suggesting that sugarcane top is a source of polyphenols with antioxidant capacity.

The aim of this study is to evaluate the effects of sugarcane top STEE on cognitive function and its potential as a novel dietary supplement, through in vivo experiments with an aging model mouse and in vitro experiments using primary human neural stem cells (hNSCs).

Firstly, the senescence-accelerated mouse prone 8 (SAMP8) mice were used as an in vivo model to assess the effect of STEE on pathological aging. SAMP8 mice develop a premature and progressive multisystemic senescence phenotype, including learning and memory impairments and pathological features similar to Alzheimer's disease (AD). SAMP8 mice may therefore be advantageous as a model between pathology and the onset of it compared to transgenic models. In this study, SAMP8 mice were randomly divided into two groups: SAMP8 water-treated group and STEE-fed group. Senescence-accelerated mouse resistant 1 (SAMR1) mice, which carry the SAM-related genotype and are resistant to accelerated aging, were also used as a reference of normal aging. STEE was administered orally at 20 mg/kg for 30 days to SMAP8 mice. In addition, the synthetic nucleoside 5-Bromo-2'-deoxyuridine (BrdU) was given to the mice through drinking water for 9 consecutive days to label the newborn cells.

After 30 days of the administration, the Morris water maze (MWM) test, a behavioral test battery assessing spatial learning and memory abilities of mammals, showed a significant improvement in escape latency from water in the SAMP8 group fed with STEE. In the test without escape platform (probe test), the STEE-fed mice increased the crossing virtual platform and the time spent in the target quadrant, compared to the SAMP8 control group. These data indicate that STEE is effective in restoring cognitive function in SAMP8 mice.

To elucidate the mechanism of the effect, brains were extracted from the mice and subjected to immunohistochemical analysis of the hippocampus, biochemical quantification of neurotransmitters, and global gene expression analysis by microarray. Immunohistochemical analysis showed an increase in newborn neurons (approximately 1.6-fold) in the hippocampus of STEE-fed mice. Protein quantification showed restoration of neurotransmitters in the cerebral cortex of STEE-fed SAMP8 mice. These data indicate that increased neurogenesis and neurotransmitter levels may be correlated in rescued age-related memory loss in SAMP8 mice. Microarray analysis revealed that STEE regulates a wide range of biological processes in the cerebral cortex of SAMP8 mice, including genes associated with neurotrophin signaling, glucose metabolism, and especially genes related to neurodevelopment (Tenm2, Tenm4, Elavl4, Ntn5, Bhlhe22, Lgr5, Msi2, etc.). These suggest a positive circulatory effect of STEE on the brain of accelerated aging. Furthermore, Mbp, Syt1, and Erc2 were upregulated in the mice cortex, suggesting enhanced synaptic plasticity by STEE.

Next, in vitro study using qRT-PCR and immunostaining with hNSCs were performed to evaluate the ability of STEE to induce NSCs differentiation. Evaluation of the pro-neurogenic potential of STEE by checking differentiation markers expression in hNSCs showed that STEE upregulated neurodifferentiation markers and downregulated stem cell markers, supporting the in vivo results. Further molecular biological analyses revealed that STEE induced neurogenesis from hNSCs by regulating the basic helix-loop-helix (bHLH) transcription factor. Interestingly, the immunocytochemical analysis showed the expanded astrocytic process in the culture with STEE, indicating promoting its maturation by the extract. These results suggest that STEE contributes to both neuronal differentiation and astrocyte morphological maturation in vitro cultures.

Further molecular biological experiments were carried out in vitro using pure compounds of STEE’s major polyphenols and hNSCs to uncover the bioactive constituents of STEE (The contents of this paragraph include materials of patent-pending and future publication. It is needed to be confidential; Relevant part: Chapter 4 of the thesis). By measuring the expression levels of neuronal differentiation markers, the potential of STEE and its major polyphenolic constituents (including their combinations) on neuronal differentiation enhancement was assessed. As a result, 3CQA, 5CQA, and ISO were predicted to be predominantly active in STEE’s pro-neurogenic effect. These components regulated cell cycle progression and cell fate determination, suggesting that they cooperatively contributed to neuronal differentiation marker upregulation by STEE. Also, STEE and its compounds stimulate mitochondrial activity and peroxisome proliferator-activated receptor- gamma coactivator-1α (PGC-1α) expression, which have been suggested to be necessary for astrocyte morphogenesis and maturation, in hNSCs-derived immature astrocytes. This result revealed the molecular basis of the effect of STEE on astrocyte morphological maturation.

In conclusion, these findings collectively suggest that STEE is a cocktail of polyphenolic compounds that have a positive effect on brain function through neurogenesis promotion. This polyphenolic cocktail modulates the differentiation of neural stem cells and astrocyte morphological maturation through their coordinated action. The bioactive effects shown could counteract the decline of neurogenesis or astrocyte morphological deficit in the aging brain and further prevent irreversible neuron and synaptic loss, which is a conventional challenge in ameliorating cognitive decline. One of the advantages of STEE is that it contains multiple bioactive compounds, and therefore may be expected to target multiple pathologies simultaneously compared to conventional drugs for age-related cognitive decline. STEE has great potential for application as a nutritional intervention or dietary supplement to improve cognitive function. Prospects for the future include the development of functional foods using this material.