Cell viability analysis using EA.hy926 cells treated with (A) ECE (0, 3, 10, 30, and 100 µg/mL) and (B) DK (0, 4, 13, 40, and 134 µM). Intracellular NO production by (C) ECE and (D) DK. Results are expressed as the mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05, *** p < 0.001 significantly different compared with the control group. EC, E. cava extract; DK, Dieckol; NO, nitric oxide; ns, not significant; -: no sample treatment (control)

Evaluation of vasodilation-related protein expression. Examples of Western blot analysis, (A,E). The protein expressions of ECE treatment on (B) phosphorylated-PI3K (p-PI3K), (C) p-Akt, and (D) p-eNOS; the protein expressions of (F) p-PI3K, (G) p-Akt, and (H) p-eNOS following DK treatment. The protein bands were ultimately developed and photographed with the FUSION Solo Vilber Lourmat system. Quantitative data were analyzed using ImageJ software. Results are expressed as the mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different compared with the control group. EC, E.Cava extract; DK, Dieckol; PI3K, phosphoinositide 3-kinases; eNOS, endothelial nitric oxide synthase; -: no sample treatment (control)

Quantification of [Ca²⁺]cytol levels following treatment with different ECE and DK concentable 926. cells. In the absence of [Ca²⁺]cytol using the Fluo-4 calcium indicator, (A) traces and (B) box plot representation of [Ca²⁺]cytol levels under ECE treatments (3, 10, 30, and 100 µg/mL); The absence of [Ca²⁺]cytol levels in cell (C) traces and (D) box plot representation of [Ca²⁺]cytol levels in response to the addition of DK (0, 4, 13, 40, and 134 µM). Results are expressed as the mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different compared with the control group. EC, E.Cava extract; DK, Dieckol; ns, not significant; AUC, area under the curve; [Ca²⁺]cytol, calcium level in the cytosol.

Computational prediction of the AchM3R and docking stimulation with DK. (A) 3D diagram and (B) 2D diagram of AchM3R and DK complex. (C) Results of the docking experiments with DK with AchM3R. DK, Dieckol; AchM3R, muscarinic M3-receptor.

Influence of a specific antagonist on [Ca²⁺]cytol levels in EA.hy926 cells treated with DK. (A) Traces and (B) box plots indicating [Ca²⁺]cytol levels in response to treatment with DK (134 μM) and antagonist. (C) Effect of DK on NO production in EA.hy926 cells pretreated with atropine. The NO levels were detected by adding 10 μM of 4 amino-5-methylamino-2′, 7′-difluorescein diacetate (DAF-FM-DA). Results are expressed as the mean ± standard deviation (SD) of three independent experiments, *** p < 0.001 significantly different compared with the control group; ### p < 0.01 significantly different compared with DK only group. DK, Dieckol; ns, not significant; AUC, area under the curve; [Ca²+]cytol, calcium level in the cytosol; NO, nitric oxide; -: no sample treatment (control)

The survival rate of the zebrafish embryos under DK treatment. The test was based on the exposure of newly fertilized zebrafish eggs to 4, 13, 40, and 134 µM of DK for up to 120 h (n = 15 per treatment, three replicates). Embryos were observed at each time point under the stereomicroscope (magnification used in the stereomicroscope for observations was 4×). DK, Dieckol.

Cardiovascular parameters measured at the dorsal aorta (DA) of 3 dpf Tg(flk:EGFP) transgenic zebrafish. (A1) Images of the DA captured by fluorescence microscope (20×) (a–e) Vasodilation observed by treatment with DK. (a) PBS (Control); (b) 4 μM of DK; (c) 13 μM of DK; (d) 40 μM of DK; (e) 134 μM of DK. (A2) Evaluation of DA vessel diameter. (B) Arterial pulse (beats per minute); (C) Mean blood flow velocity (μM/s); (D) Blood flow (nL/s) were measured from n = 6 larvae per group. The yellow box represented the area used for evaluation. Results are expressed as the mean ± standard deviation (S.D.). * p < 0.05, ** p < 0.01. ## p < 0.01. *** p < 0.001 significantly different compared with the control group. dpf, days post-fertilization; ns, not significant; -: no sample treatment (control)

A schematic represented the potential endothelial-dependent vasodilation mechanisms of DK. DK possess potential vasodilatory effects by upregulating the expression of PI3K/Akt/eNOS and enhancing the [Ca²⁺]_cytol transit via AchM3R, resulting in NO formation in endothelial cells.