Assessment of neuroprotective activity of hydroxytyrosol rich extracts
Assessment of neuroprotective activity of hydroxytyrosol rich extracts from olive oil
Background
Oxidative processes in the brain are believed to play a crucial role in the development of age-related neurodegenerative diseases. Several strategies can be used to overcome these increasing numbers. There are epidemiological studies supporting a negative correlation between fruit and legumes consumption and neurodegenerative incidence, being the phytochemicals associated to these beneficial effects mainly antioxidant compounds. Products yet to be biologically characterized, such as hydroxytyrosol (HT)-rich extracts from olive oil, represent a novel pool of phytochemicals that can provide and potentiate future bioactive principles for neurodegenerative diseases prevention and/or therapy. A neuroblastoma cell line will be challenged by oxidative stress (hydrogen peroxide), a condition associated to neurodegenerative diseases. In order to evaluate the neuroprotective activity of HT rich extracts, important oxidative stress markers will be cross compared in treated or non-treated cells.
This proposal is part of a major project that intends to develop new bioactive agents for the prevention and/or therapy of age-related neurodegenerative diseases.
Working plan
The work will encompass the following tasks:
TASK 1: Evaluation of neuroprotective activity of extracts on oxidative-injured neuroblastoma
The cells will be treated with 300 microM hydrogen peroxide for 24h, after pre-incubation with HT-rich extract for 24h, at non-toxic concentrations already defined in host lab. Cell viability will be assessed through flow cytometry/ fluorescent microscopy using PI and Dioc 6 (3).
TASK 2: Assessment of cell death/survival mechanism
Acute and chronic neurodegenerative conditions have been associated with both apoptotic and necrotic cell death, and caspases can play a role in both mechanisms. Caspase 3 and 7 activation will be monitored by enzymatic assays and PI/Annexin V conjugates will be used as probes for NECROSIS and APOPTOSIS evaluation by flow cytometry.
TASK 3: Redox metabolic alterations
Intracellular ROS formation by fluorimetry using dichlorodihydrofluoresceindiacetate
(H2DCFDA) for hydrogen peroxide and hydroethidine for superoxide anion. Other redox metabolic alterations such as a) total thiols content (GSH/GSSG levels) will be evaluated by HPLC with fluorimetric detection. Compounds related to the glutathione metabolism will be used to reveal the importance of this antioxidant system and redox cell response.
Supervisors
Cláudia Nunes dos Santos (csantos@itqb.unl.pt)
Ana Matias (amatias@itqb.unl.pt)
Laboratory/Institution
Disease and Stress Biology Laboratory @ ITQB
Nutraceuticals and Controlled Delivery @ ITQB/IBET
