Mechanisms of Thai medicinal plant extracts on the attenuation of glutamate-mediated neurotoxicity

Abstract

As the world population ages, neurodegenerative diseases, particularly Alzheimer’s disease, are becoming a major public health concern with increasing incidence and prevalence worldwide while no cure currently exists. To challenge this situation, herbal medicine may provide a potential alternative treatment as use of natural-derived substances has been proven to be effective in the prevention and/or treatment of several diseases. During the past decades, increasing evidence has implicated excessive glutamate levels in the pathway of neuronal cell death. Thailand is a place known for cultivating a variety of tropical plants and herbs, so far many of them have never been examined for their benefits in neuroprotection. Therefore, this study aimed to investigate the effects of Thai medicinal plant extracts and their underlying mechanisms on the attenuation of glutamate-induced neuronal cell death in the mechanism of oxidative glutamate toxicity using hippocampal neuronal HT-22 cell line as in vitro model of neurodegeneration. We selected five Thai medicinal plant species including Morinda citrifolia, Caesalpinia mimosoides, Murdannia loriformis, Acanthus ebracteatus, and Streblus asper. Then the cell viability, apoptosis, radical scavenging and DCFH-DA assays were performed to assess the protective effects of plant extracts against glutamate-induced oxidative toxicity in HT-22 cells. To elucidate the underlying mechanisms, cells treated with plant extracts were analyzed for the expressions of mRNA and proteins interested by immunofluorescent staining, western blot analysis and quantitative real-time reverse transcription polymerase chain reaction techniques. The longevity effect of plant extracts was examined on C.elegans by lifespan assay. Phytochemical analysis and separation using Acid-Base extraction were performed to clarify putative phytochemical components of the promising extracts. Here, among five plant species, A. ebracteatus and S. asper exhibited profound neuroprotective properties. We demonstrate that the ethanol leaf extracts of both plant species are capable of attenuating the oxidative stress and HT-22 cell death induced by glutamate in a concentration-dependent manner. Co-treatment of glutamate with both extracts significantly reduced apoptotic cell death via inhibition of apoptotic-inducing factor nuclear translocation. The increases in nuclear factor erythroid 2–related factor 2 (Nrf2) levels in the nucleus and gene expression levels of antioxidant-related downstream genes under Nrf2 control were found to be significant in cells treated with both plant extracts. Furthermore, the extract of S. asper was able to extend the lifespan of C.elegans. Phytochemical analysis of chemical components proposed at least two molecules of interest include verbascoside from A. ebracteatus and carnosic acid from S. asper, which are possibly responsible for their neuroprotective properties. Further isolation of S. asper ethanol leaf extract into three fractions showed that the antioxidant activities were found in the order of acidic > basic > neutral, whereas the decreasing order of neuroprotective activity was neutral > basic > acidic. Moreover, TLC bioautography revealed one component in the neutral fraction exhibited anti- acetylcholinesterase (AChE) activity. While in the acid fraction, two components showed inhibitory activity against AChE. Taken together, the overall findings have supported the potential of A. ebracteatus and S. asper leaf as promising natural sources for neuroprotective agents, anti-aging agents, and/or AChE inhibitors.