The ethanol extracts of Gracilaria lemaneiformis that have inhibitory effects on Karenia mikimotoi and Skeletonema costatum were separated by liquid-liquid extraction using different polar solvents into five fractions with antialgal activities (petroleum ether, chloroform, ethyl acetate, n-butanol, and water-soluble fractions). These fractions were chromatographed on silica gel to give, after repeated preparative thin-layer chromatography (PTLC) purification processes, 1-β-D-ribofuranosyluracil (1), 3-hydroxymethyl-pyrrolopiperazine-2,5-dione (2), benzene-1,2-propanoic acid (3), 1-O-palmitoyl-2-O-palmitoleoyl-3-O-β-D-galactopyranosyl glycerol (4), 7-oxabicyclo[4.1.0]-heptan-3-ol (5), linoleic acid (6), 3,4-dimethoxy-6-(methoxymethyl)-tetrahydro-2H-pyran-2,5-diol (7), and 3,7,11,16-tetramethyl -2-heptadecen-1-ol (8). Five of them, natural products 1, 2, 5, 7, and 8, were isolated from Gracilaria lemaneiformis for the first time, and three natural products (3, 5, and 8) were isolated from marine macroalgae for the first time. Among them, natural products (1, 2, 3, 4, and 6) showed the most obvious inhibition activities to the growth of Karenia mikimotoi and Skeletonema costatum at the concentration of 80 μg/mL. Therefore, antialgal activities of these five natural products against Amphidinium carterae, Heterosigma akashiwo, Karenia mikimotoi, Phaeocystis globosa, Prorocentrum donghaiense, and Skeletonema costatum were further tested at different concentrations (0.4, 2, 10, and 50 μg/mL). This was the first report of antialgal activities of five natural products (1, 2, 3, 4, and 6) to these six red tide microalgae. They showed significantly selective antialgal activities against all tested red tide microalgae. At the concentration of 50 μg/mL, the growth of Amphidinium carterae, Heterosigma akashiwo, Karenia mikimotoi, and Phaeocystis globosa was obviously inhibited; for Karenia mikimotoi, natural products 1, 2, and 6 have significant antialgal activities; the growth inhibition of Skeletonema costatum that was exposed to natural products 1, 3, and 4 was remarkable. Furthermore, by analyzing and comparing EC50-96 h values, it has been determined that natural product 3 (natural product 4) showed the superior application potential than potassium dichromate and some reported natural products (such as gossonorol isolated from Porphyra yezoensis, trehalose purified from Ulva pertusa) as a characteristic antialgal agent against Amphidinium carterae (Phaeocystis globosa). In addition, natural products 1 and 3 also showed good superiority than some reported natural products in inhibiting Skeletonema costatum; however, it was a pity that they were inferior to potassium dichromate in the inhibiting this red tide microalgae. Taken together, it is not hard to conclude that Gracilaria lemaneiformis was a good source of natural products with antialgal activities against some red tide microalgae.
Keywords: Antialgal activity evaluation; EC50; Gracilaria lemaneiformis; isolation; natural products; purification; red tide microalgae.