Validation of Folin – Ciocalteu assay for phlorotannins analysis in fresh and storm-cast brown algae Ascophyllum nodosum (Phaeophyceae)

Introduction. Phlorotannins are secondary metabolites produced mainly by brown seaweeds and belong to the class of polyphenolic compounds with diverse bioactivities. Storm-cast brown algae, a problem for coastal biocenoses, may be a valuable source of polyphenols. The Folin-Ciocalteu reagent (FCR) is the most commonly used for the quantification of total polyphenols in natural samples. Different spectrophotometric methods with FCR for the determination of phlorotannins in algae have been described in the literature.

Aim. The primary aim of this study is to standardize and validate the spec-trophotometric determination of total phlorotannins using FCR and demonstrate its applicability to analysis of storm-cast and fresh algae.

Materials and methods. A. nodosum samples were collected in sheltered beach on the Olenitsa Bay (66°27'15.7"N 35°18'20.4" E), Kandalaksha Gulf (White Sea, Russia) on two tidal levels: one located at low tide at a depth of 0.6–1.0 m (fresh) and the second was located at the supralittoral in the zone of wave splashing (storm-cast). Field sampling was carried out between June and September. The cleaned seaweed were transported to the laboratory, washed accu-rately with clean water, freeze-dried, ground into powder. Functional groups present in the algae were identified using Fourier Transform Infrared (FT-IR) spectroscopy. Spectrophotometric determination of total phlorotannins content (TPhC) with FCR was used and validated according to national and international guidelines.

Results and discussion. The optimum conditions for analysis time, wave-length, and standard substance were 45 min, 750 nm, and phloroglucinol, respec-tively. Under these conditions, validation by UV/Vis spectrophotometry proved the method to be linear (R² > 0.99), specific, precise, accurate, reproducible, robust, and easy to perform. The limit of detection and limit of quantification were 0.005 and 0.02 mg/mL, respectively. For precision analysis, an intra-day test (RSD 2.16 %) and an inter-day test (RSD 2.84 %) were performed. Matrix effect assessment demonstrated that this had a negligible effect (1.9 %) on the phlorotannins quantification. TPhC in storm-cast algae ranged from 59 to 101 mg/g, while freshly collected algae were statistically significantly higher (p < 0.01) and ranged from 71 to 135 mg/g. Maximum accumulation of phlorotannins in A. nodosum was observed between July and August, after which a decrease was observed.

Conclusion. Results of current study could be utilised for routine analysis of TPhC in brown algae and storm-cast seaweed using optimized spectrophotometric method with FCR on readily available low-cost equipment in most laboratories to provide rapid. This methodology complies with the requirements for pharmaceutical analysis to ensure the reliability of results during pharmaceutical development and routine control both in fresh and storm-cast of A. nodosum.

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