The cyanobacterium Nostoc sp. and an intertidal cyanobacterial mat community as a source of scytonemin and dihydroscytonemin

Abstract

The cyanobacterium Nostoc sp. Strain BEA1032B was isolated by the Banco Espan˜ol de Algas (BEA) from a biofilm on the road to El Hornillo (Gran Canaria) and it attracted our attention in the project Desarrollo Industrial de Canarias (DISCan-2007) as a potential source of high added value products. The antecedents pointed out the presence of a high content of oxidized scytonemin (Scy) and reduced scytonemin (H2Scy) in the organic extracts of Nostoc spp. The oxidized compound (Scy) is an inhibitor of kinases key in hyperproliferative inflammatory diseases. The reduced compound (H2Scy) induces autophagic cell death in human T-lymphoid cell line Jurkat cells. With these precedents, in this study we have decided to deepen into the chemical, biochemical and pharmacological knowledge of some cyanobacteria from the coast of Canary Islands. The cyanobacterium Nostoc sp. Strain BEA1032B was cultivated at 20 ºC in a growth chamber under a photon irradiance of 100 µmol m−2 s−1. The cyanobacterial mats were collected from an intertidal flat in Las Palmas de Gran Canaria (Canary Islands). The cyanobacterium Nostoc sp. Strain BEA1032B and the cyanobacterial mats were extracted in a Soxhlet apparatus with acetone followed by rotary evaporation concentration, a brown extract was obtained. This extract was fractionated by silica gel column chro- matography eluted with n-hexane/ethyl acetate mixtures of increasing polarity, and successive fractions were analyzed by thin-layer chromatography (TLC) eluting chloroform-methanol (9:1). The cytotoxic activity assays were performed by Bioqu´ımica Farmacolo´gica group of the ULPGC. Study of less polar product.- When eluted with chloroform-methanol (9:1) on TLC (silica gel) it shows a green spot at Rf = 0.40. It was identified as scytonemin (Scy) by 1H-NMR spectra, both in one and two dimensions. Study of the more polar product.- When eluted with chloroform-methanol (9:1) on TLC (silica gel) it shows a red spot at Rf = 0.35. It was identified as dihydroscytonemin (H2Scy) by 1H-NMR spectrum. HPLC analysis.- Both metabolites were separated by HPLC using a column Waters µBondapak C18, according to the following elution program: 80% H20/ 20% MeOH for 2 min; then 13 min to reach 100% of MeOH by linear gradient. Flow rate was 1 ml min−1. UV detection was set at 386 nm and with these conditions scytonemin (Scy) and dihydroscytonemin (H2Scy) were eluted at 14.48 and 15.55 minutes, respectively. Cytotoxic activity.- Both products showed a cytotoxic activity against the human leukemia cell line HL- 60 IC50 (Scy) = 4.6 µM; IC50 (H2Scy) = 1.9 µM. Similar results were found against the human leukemia cell line U937. As conclusion, Nostoc sp. Strain BEA1032B and the intertidal cyanobacterial mat community are good raw materials to obtain scytonemin (Scy) and dihydroscytonemin (H2Scy). Both products showed cytotoxic activity against human leukemia cell lines, HL-60 and U937.