Chemical And Biological Studies On Neuritogenic Substance From Traditional Chinese Medicine

Alzheimer's disease (AD) is the most common neurodegenerative disorder among the elder population. Current approaches for the treatment of AD provide temporary symptomatic relief and do not cure the disease. It is urgently demanded to develop a new strategy for treating AD. It was reported that nerve growth factor (NGF) was considered as a candidate for the treatment of AD. NGF plays a key role in stimulation, survival and functional maintenance of the neurons. However, NGF is a large molecule and unable to cross the BBB. The application of NGF as a therapeutic agent is assumed to be difficult. Therefore, more attention has focused on finding the small molecules that have the neurotrophic properties of NGF and be able to cross the blood-brain barrier.After screening more than one hundred extracts from traditional Chinese medicine with the PC 12 cell line, we found that the extracts of Ophiopogon japonicus (Thunb.) Ker-Gawl and Mimosa pudica have significant neuritogenic activity. The active extracts were chromtographed on silica gel and ODS, and then purified by repeated HPLC to afforded twenty-three active compounds, guided by bioassay. In addition, the active fractions isolated from the edible Chinese mushroom Termitomyces albuminosus (Berk.) Heim. afforded two neuritogenic compounds. The chemical structures of twenty-one compounds were determined by spectroscopic analysis and comparison spectroscopic data with those reported. We have isolated seventeen steroidal saponins from O. japonicus; two cerebrosides from T. albuminosus and two unsaturated fatty acids from Mimosa pudica. Among them, six compounds possess new chemical structures. The structures of several active compounds are under the elucidation due to their amount and complex structures.All the steroidal saponins exhibited the neuritogenic activity against PC 12 cells. Ophiopogonin D, which is the highest content among them, has the best neuritogenic activity. To study the structure-activity relationships and find a lead coumound, ophiopogonin D was hydrolyzed to give the aglycone, ruscogenin. The aglycone showed similar neuritogenic activity compared with ophiopogonin D. The result revealed that steroidal saponins have the neuritogenic activity despite the difference of sugar moieties. To explore the mechanism of action induced by steroidal saponins on PC 12 cells, specific inhibitors were applied for finding possible related kinases. The result showed that the percentage of neurite outgrowth induced by ophiopogonin D significantly decrease, when treated with LY294002 and PD98059. Therefore, the neuritogenic activity induced by ophiopogonin D was depentent on the activation of PI3K and MEK/ERK signalling pathway. In addition, our previous work showed that steroids skeleton played a key role in their anti-aging activity, based on the yeast bioassay. Thus, the anti-aging effects and mechanisms of steroidal saponins in yeast were investigated. The results suggested that SOD1, SOD2 and uth1 genes were assumed to play an important role in the anti-aging effects in yeast.Further examination of active fractions from T. albuminosus resulted in the isolation of two neuritogenic compounds. Their chemical structures possessed a unique modification by a hydroxyl group at C-9 of the LCB moiety. Subsequently, the biological results showed that the chain length of fatty acyl group of cerebrosides played a key role in the neuritogenic activity. To investigate the signalling pathway induced by cerebrosides, the specific-inhibitor experiments were done to find the possible target kinases. The result revealed that significant neuritogenesis induced by cerecrosides was related with the PI3K, MEK/ERK and PKC signalling pathway.In our studies on searching for neuritogenic substances using the PC 12 cell line, a series of steroidal saponins, cerebrosides and unsaturated fatty acids were isolated from the traditional Chinese medicine and the edible Chinese mushroom, which exhibited the neuritogenic activity. The possible signaling pathway of these active compounds was investigated and finally found the related target kinases. Intensive studies on these neuritogenic compounds are currently underway for the treatment of Alzheimer's disease.