ACTIVITY 4

The antioxidant capacity of the extracts and essential oils will be measured via standard spectrophotometric methods: DPPH, ABTS and FRAP, CUPRAC and lipid peroxidation inhibition. As each method has its advantages and disadvantages and not all are highly related, the antioxidant capacity should be evaluated by comparison between the adopted methods.

The plant extracts and essential oils obtained, in activity 3 will be assessed as to their antioxidant and anti-inflammatory abilities, namely regarding inhibition of ROS generation and NO production, using fluorescent probes. Moreover, the expression of inflammatory inducible enzymes such as nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 will be evaluated in human cell lines (cancer and non-neoplastic), by immunocytochemistry.

In vitro evaluation of the anti-proliferative and cytotoxic activity of the aqueous extracts will be performed for human cancer cells, by standard assays of growth-inhibition and viability determination: e.g. BrdU, Sulphorodamine B, MTT (formazan-based) or Alamar (resazurin-based) colourimetric tests; and LDH leakage for cell death quantification.

Several parameters will be investigated:

1. concentration within a range suitable for future applications;
2. incubation time;
3. type of cell line.

Different human cell lines will be tested, namely breast cancer and skin melanoma. Non-neoplastic cells will also be used, as controls. This activity will be carried out at ESAC/IPC, QFM-UC and REQUIMTE.

With these experiments, we will screen the potential antioxidant, anti-inflammatory ability and interference with cancer cell proliferation of extracts from halophytes plants and maritime plant corema.