(77e) Inhibitory Effects of Microalgal Extracts and Their Active Compounds On Protein Glycation and Glycoxidation

The Maillard reaction is a nonenzymatic reaction between reducing sugars and the free amino groups of proteins that leads to the formation of advanced glycation end products (AGEs). The glycation causes gradual deterioration in the structure and function of tissue proteins, and it is believed that the accumulation of AGEs acts as a major pathogenic process in diabetic complications and many other health disorders.

In our study, the antiglycative capacities of 20 microalgae at different growth phases were evaluated for the first time. In a bovine serum albumin (BSA)-glucose system, ethyl acetate fractions of green microalgae Chlorella and diatom Nitzschia laevis exhibited the highest inhibitory effects against the formation of total AGEs (inhibition rates: 81.76% - 88.02% and 91.68%) at a concentration of 500 ppm. Such abilities were higher than the effect of 1 mM aminoguanidine (AG) solution (inhibition rate: 80.51%), a commonly used inhibitor of glycation process. The band intensity of these proteins was compared on the SDS-PAGE and calculated. In addition to total AGEs, these fractions were also found to be effective in the blockage of the formation of two specific AGEs, pentosidine and N-carboxymethyllysine (CML) by measuring the typical fluorescence and an enzyme-linked immunosorbent assay (ELISA).

Microalgal extracts with strong glycation inhibitory activity also contained antioxidant activity and chelating properties when a DPPH radical scavenging assay and Trolox equivalent antioxidant capacity (TEAC) assay as well as metal chelating assay were applied. We further demonstrated that ethyl acetate fractions of Chlorella and Nitzschia laevis decreased the amount of hydroxyl radicals in the benzoate hydroxylation, indicating their preventative roles in sugar autoxidation. Moreover, these fractions were proved to be effective in the prevention of oxidative protein damage by inhibiting the protein carbonyl formation and the loss of thiol groups in a time-dependent manner, which are believed to occur under the glycoxidation processes.

The cell model studies were also performed. The treatment of AGEs resulted in the proliferation of human retinal epithelial (RPE) cells, which was probably associated with the development of diabetic retinopathy. Using the tetrazolium dye-reduction (MTT) assay, we found ethyl acetate fractions of Chlorella and Nitzschia laevis significantly suppressed this proliferation. In addition, confirmed by both RT-PCR and ELISA, these fractions inhibited the upregulation of vascular endothelial cell growth factor (VEGF) induced by AGEs. Decreased intracellular generation of reactive oxygen species (ROS) was also observed using a confocal laser scanning microscopy (CLSM).

Subsequent HPLC and gas chromatography (GC) analyses revealed that carotenoids in Chlorella and polyunsaturated fatty acids, mainly of linoleic acid, arachidonic acid and eicosapentaenoic acid in Nitzschia laevis contributed to their strong antiglycative capacities, suggesting their beneficial roles in the protection against diabetic complications.

In summary, a relatively large number of microalgal samples were systematically evaluated for their inhibitory capacities on protein glycation and glycosidation. Using both cell-free and cell models, ethyl acetate fractions of the green microalgae Chlorella and diatom Nitzschia laevis exhibited significant inhibitory effects against the formation of AGEs. Since the accumulation of AGEs is a major pathogenic process of diabetic complications, these results support that microalgae are beneficial food and preventive agent choices for diabetics. Carotenoids in Chlorella and polyunsaturated fatty acids, mainly of linoleic acid, arachidonic acid and eicosapentaenoic acid in Nitzschia laevis were found to contribute to their strong antiglycative capacities. They are essential nutritional components for human health.