Analysis of polyphenolic content in marine and aquatic angiosperms from Norwegian coastal waters
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In this work, the polyphenolic content in extracts of Zostera marina L., Zostera noltii Hornemann, Ruppia cirrhosa (Petagna) Grande and Ruppia maritima L. from Norwegian coastal waters was characterized for the first time. In Z. marina and Z. noltii fifteen different flavones, as well as rosmarinic acid were identified. Eight of the flavones were found to be sulphated, among these were luteolin 7,3'-O-disulphate and chrysoeriol 7-O-sulphate – structures previously not published with complete NMR assignments. In addition, minor amounts of luteolin 7-O-β-(6''-Omalonyl) glucopyranoside (6) and apigenin 7-O-β-(6''-O-malonyl)glucopyranoside (11) were identified in Z. marina and Z. noltii for the first time. The sulphated flavones were stable in neutral and slightly acidic (< 0.1% trifluoroacetic acid) extracts, but quickly decayed to their corresponding aglycones under more acidic conditions (≥ 0.5% trifluoroacetic acid). Moreover, purified flavonoid sulphates often decomposed during the final steps of isolation, due to increased acid concentrations when the solvents were removed by rotary evaporation. In R. cirrhosa and R. maritima eight flavonoids were identified, namely the 3-O-glucopyranosides and 3-O-galactopyranosides, as well as malonylated 3-O-glycosides of quercetin and isorhamnetin. The main compound in both species was chicoric acid. None of these compounds have been found in either Ruppia species before.
Individual and total phenolic content was quantified in crude extracts of all four seagrass species using analytical HPLC with UV-Vis detection. The flavonoid content was 18.1‒24.5 mg/g (DW) in Z. marina and 26.2‒30.5 mg/g (DW) in most of the examined Z. noltii populations. Yet, Z. noltii plants collected at the localities Gripnesvågen (C) and Huglo (D), which are in proximity to each other, contained the highest (34.3 mg/g) and lowest (17.3 mg/g) flavonoid concentrations, respectively. The flavonoid content was generally lower in R. cirrhosa and R. maritima than in the Zostera species. However, the phenolic acid content was remarkable high in Ruppia, with chicoric acid concentrations in the range of 11.1‒12.7 mg/g in R. cirrhosa and 27.9‒ 30.2 mg/g in R. maritima. The flavonoid content in the three R. cirrhosa populations from different localities on the West coast differed significantly, with flavonoid concentrations ranging from 5.9 mg/g to 14.7 mg/g.
Seasonal variation of both flavonoids and phenolic acids in Z. marina, Z. noltii and R. cirrhosa was examined. The quantitative variation of flavonoids and rosmarinic acid was found to be relatively consistent from year to year in Z. marina during a period of three years. The two Zostera species did appear to have a different flavonoid production in the various seasons. While Z. marina had the highest content in young leaves in May or June and lowest in February, the opposite was observed in Z. noltii, with lowest flavonoid content in May/June and highest in February. The variation of flavonoid content in R. cirrhosa appeared to follow a similar pattern as the one observed in Z. marina, with the highest concentration of flavonoids in summer (August). However, while the concentrations of rosmarinic acid were highest in late spring/early summer (May/June) in Z. marina (3.6 mg/g), peak concentration of chicoric acid was observed in March in R. cirrhosa (29.2 mg/g).
The antioxidant activity of Ruppia cirrhosa extracts and isolated compounds was investigated spectrophotometrically by a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. IC50 values were 152.9–175.7 μg/mL for Ruppia cirrhosa crude extracts, which is considered low radical scavenging activity. However, a partially purified R. cirrhosa extract exhibited very strong radical scavenging activity, with an IC50 value of 31.8 ± 0.7 μg/mL. IC50 values for isolated flavonoids ranged from 12.1– 88.4 μg/mL.