Zostera marina L. was collected during spring low tide by hand at a locality close to Espegrend Marine Biological Station outside Bergen, Norway. The sample locality (60˚16'12.0''N, 05˚13'20.3''E) was situated in a small sheltered bay, influenced by fresh water from a small brook. Z. marina formed a large patch growing in fine, muddy sediment. The collected material was washed thoroughly in fresh water and air-dried. The root was separated from the rest of the plant, and the material was cut in small pieces and stored at −20˚C, when not used. A voucher specimen has been deposited in the Herbarium BG at the University Museum of Bergen, Bergen.

The seagrass was extracted 3 times with 50% aqueous methanol, after optimization of extraction conditions. The extracts were filtered through glass wool, and the methanol was removed using a rotary evaporator under reduced pressure at 27˚C, followed by partitioning with ethyl acetate. The aqueous layer, containing the flavonoids, was further concentrated and applied to an Amberlite XAD-7 column (70 × 5 cm, Sigma-Aldrich, Steinheim, Germany). The flavonoids were eluted with distilled water until the fractions were colorless, and then methanol was applied for elution of adsorbed flavonoids. Obtained fractions were analyzed by analytical HPLC-DAD, and fractions containing similar qualitative flavonoid content were combined and concentrated under reduced pressure. The semi-purified plant extract was submitted to preparative HPLC to obtain purified compounds. The purified fractions were evaporated under reduced pressure at 27˚C, and were further analyzed by HRLC-MS and NMR spectroscopy.

Approximately 50 mg of dried Z. marina leaves was extracted with 50% methanol with 0.1%, 1.0% formic acid, 0.1% and 0.5% trifluoroacetic acid (TFA) for 1 hour at 25˚C. The extracts were filtered and analyzed periodically by analytical HPLC over 3 months period, and compared with a corresponding extract containing no acid. The relative content of sulphated flavonoids in the extract was determined by peak area measurement at 360 nm of individual compounds, relative to the total area of all flavonoids in the sample.

Analytical HPLC: The Agilent 1100 HPLC system was equipped with a HP 1050 diode array detector and a 200 × 4.6 mm inside diameter, 5 μm ODS Hypersil column (Supelco, Bellefonte, PA). Two solvents, (A) water (0.5% TFA) and (B) acetonitrile (0.5% TFA), were used for elution. The elution profile for HPLC consisted of initial conditions with 90% A and 10% B followed by a linear gradient elution to 50% B. The flow rate was 1.0 mL/min, and aliquots of 15 μL were injected with an Agilent 1100 series microautosampler. The UV-Vis absorption spectra were recorded online during HPLC analysis over the wavelength range of 240 - 600 nm in steps of 2 nm. Preparative HPLC: The system used a Gilson 321 pump equipped with an Ultimate 3000 variable wavelength detector, a 25 × 2.2 cm (10 μm) Econosphere C18 column (Grace, Deerfield, IL), and the solvents (A) water (0.1% formic acid) and (B) acetonitrile (0.1% formic acid). Following gradient was used: 0 - 5 min; 15% - 20% B, 5 - 25 min; 20% - 30% B, 25 - 28 min; 30% - 40% B, 28 - 30 min 40% - 15% B. The flow rate was 15 mL/min. NMR-spectroscopy: One-dimensional ¹H, 2D heteronuclear single quantum coherence (¹H-¹³C HSQC), heteronuclear multiple bond correlation (¹H-¹³C HMBC), double quantum filtered correlation (¹H-¹H DQF COSY) and total correlation spectroscopy (¹H-¹H TOCSY) experiments were obtained on a Bruker 600 MHz instrument equipped with a cryogenic probe. Sample temperatures were stabilized at 298 K. The deuteriomethyl¹³C signal and the residual ¹H signal of the solvent (d₆-DMSO) were used as secondary references (δ 39.5 and 2.5 from TMS, respectively).High-resolution LC-electrospray mass spectrometry (ESI⁺/TOF), spectra were recorded using a JEOL AccuTOF JMS-T100LC in combination with an Agilent Technologies 1200 Series HPLC system at the following instrumental settings/conditions; Ionization mode: positive, ion source temperature = 250˚C, needle voltage = 2000 V, desolvation gas flow = 2.0 L/min, nebulizing gas flow = 1.0 L/min, orifice1 temperature = 100˚C, orifice2 voltage = 6 V, ring lens voltage = 18 V, ion guide peak voltage = 2000 V, detector voltage = 2300 V, acquisition range = 100 - 1000 m/z, spectral recording interval = 0.5 s, wait time = 0.03 ns and data sampling interval = 0.5 ns. Sample was solved in a mixture of water and acetonitrile with 0.1% formic or acetic acid. The elution profile for HPLC consisted of initial conditions with 90% A (water with 0.1% formic acid) and 10% B (acetonitrile with 0.1% formic acid), isocratic elution 0 - 2 min, followed by a linear gradient elution to 50% B (2 - 15 min). A 50 × 4.6 mm internal diameter, 1.8 μm Agilent Zorbax Eclipse XDB C18 column was used for separation.

The HPLC profile of Zostera marina extract (Figure 1) revealed the presence of three major (1, 4, 8) and five minor flavones (2, 3, 5, 7, 9) (Figure 2 and Table 1), together with higher amounts of rosmarinic acid (6). In addition, traces of six flavones (10-15) were found during HRLC-MS examinations of the extracts. Five of these flavones (1, 2, 4, 7, 8) were substituted with sulphate groups, and the order of retention times in the HPLC reversed phase column system was found to be: disulphate (1) < monoglucoside (3) < monosulphate (4) < acyl glucoside (5) < aglycone (9), here exemplified with luteolin derivatives.

As shown in Figure 3 the UV absorption spectra of luteolin 7-sulphate (4) and luteolin (9) are relative similar, and their UV_max values are consistent with previously reported data for flavones and flavone glycosides [22] , whilst the significant hypsochromic shift in the UV_max of luteolin 7,3'-disulphate (1), is strongly indicating the presence of a sulphate group in the 3'- or 4'-position on the B-ring. Thus, introducing a sulphate group to the flavonoid A-ring, does not influence the UV absorption significantly, but sulphation in the 3'- or 4'-position on the B-ring will cause a large hypsochromic shift in band I. Thus flavonoid sulphates seem to have analogous UV spectral characteristics as their corresponding flavonoid glycosides [12] .

The stability of the sulphated flavones in Z. marina extracts was investigated under various acidic conditions. The compounds were quite stable in extracts containing 0.1% - 1.0% formic acid and in 0.1% TFA, and did not

sh = shoulder. ^*only found in trace amounts in extracts (10: apigenin 7-glucoside, 11: apigenin 7: (malonyl)glucoside, 12: diosmetin- or chrysoeriol 7: (malonyl)glucoside, 13: apigenin, 14: chrysoeriol, 15: diosmetin).

show significant differences when compared to their storage in the corresponding neutral methanolic extract for 3 months. However, in the extract containing 0.5% TFA, the flavone sulphates (1, 2, 4, 7 and 8) decomposed gradually to their corresponding aglycones (9, 14 and 15) due to acid hydrolysis. The sulphated flavones were isolated and purified by preparative HPLC, and their stability in the eluate solvent (consisting of water and acetonitrile with 0.1% formic acid) were monitored by analytical HPLC. The results showed that the sulphated flavones were relative stable in this solvent with a decay of 1% - 5% in the course of 10 days. However, when the solvent was removed by evaporation, these compounds quickly decomposed to their corresponding aglycones, due to accumulated acid concentrations. Despite the problems with instability of the sulphated flavones, we were able to obtain pure samples of 1 (14 mg), 4 (4 mg) and 7 (6 mg).

The ¹H NMR spectrum of compound 1 (Figure 2) showed six proton signals in the aromatic region; a pair of meta coupled protons at δ 6.57 (1 H, d, J = 2.06 Hz, H-6) and δ 6.98 (1 H, d, J = 2.01 Hz, H-8), a one proton singlet at δ 6.74 (H-3), and the AMX system at δ 6.99 (1 H, d, J = 8.3 Hz, H-5'), δ 7.93 (1 H, d, J = 2.34 Hz, H-2'), δ 7.71 (1 H, d, J = 2.35, 8.7 Hz, H-6'), which were in accordance with a luteolin derivative [23] . The ¹³C NMR values for compound 1 (Table 2) were assigned on the basis of ¹J_CH, ²J_CH, ³J_CH and ⁴J_CH correlations observed in the HSQC and HMBC spectra. The downfield carbon data for C-6, C-8 as well as the significantly downfield shifts of H-6 and H-8 strongly indicated the presence of an electron withdrawing sulphate ester in position C-7. Similarly, a second sulphate group was indicated by the NMR values of the protons H-2', H-5' and