We measured the free phenolics content of the seagrass leaves of the different species. Seagrass samples for this analysis were taken in nearby meadows in Snake Island, an island approximately 1 km away from Changuu Island. We took three seagrass leaf samples in three meadows of each seagrass species (T. ciliatum, T. hemprichii, H. uninervis, S. isoetifolium, and C. serrulata) and transported them in a cold container to the laboratory in the Institute of Marine Sciences (Stone Town, Zanzibar).
We cleaned the leaves from epiphytes and rinsed them with distilled water. The cleaned leaves were dried in an oven at 38°C for ~3 days and transported to the Leibniz Centre for Tropical Marine Research in Bremen (Germany). The leaves were crushed to a fine powder using a mortar and pestle with liquid nitrogen, and the content of free phenolic compounds was measured using photometric determination of the phenolic compounds as tannic acid equivalents, following a modified protocol by Ainsworth and Gillespie (2007). To create the calibration curve, we prepared a standard tannic acid solution and transferred it to Eppendorf tubes in increasing volumes (from 0 mL to 0.450 mL in 0.050 mL intervals) filling up to a volume of 0.5 mL with 70% Ethanol. We extracted 10 mg of dried sampled with 2 mL of 70% Ethanol for 24 hours on a shaker at 225 rpm and centrifuged them for 10-20 minutes at 10000g-20000g (until the supernatants were clear). An aliquot of the supernatant (between 0.02 and 0.1 mL) was transferred to 2 mL Eppendorf tubes, filling up to a volume of 0.5 mL with distilled water. We added 1250 µL of Na2CO3-NaOH-solution (0.7 N Na2CO3 and 0.1 N NaOH) to standards and samples. We added 250 µL 1N Folin-Ciocalteu reagent and placed 340 µL of the standards and samples into a 96er well plate in the dark, and measured after 2h with TECAN Infinite M200 Pro plate reader at 760 nm.
The concentration of tannic acid equivalents in the samples was calculated following the calibration curve, by multiplying the concentration of tannic acid equivalent to the factor of volume extract containing 1 g of leaf powder and the dilution factor of extract used. Final results are shown in miligrams of free phenolics as tannic acid equivalents per gram of seagrass dry weight.
