Zostera marina, L.

Li, Yan, R, Lucie, Scarpato, Silvia, Çiçek, Serhat Sezai, Jordheim, Monica, Stenclov, Tereza, Strnad, Miroslav, Mangoni, Alfonso & Zidorn, Christian, 2022, Seasonal variation of phenolic compounds in Zostera marina (Zosteraceae) from the Baltic Sea, Phytochemistry (113099) 196, pp. 1-10 : 5-8

2.4. Chemotypes in Zostera marina

The chemosystematic/chemophenetic value of flavonoids has been recognized in terrestrial plants since comparative phytochemical investigations had been performed ( Harborne, 1965; Bohm, 1987; Zidorn, 2019). Subsequently, the flavonoid regulatory and structural pathways were characterized in detail ( Hichri et al., 2011; Huang et al., 2015). Also in Zostera flavonoids have received considerable attention ( Grignon-Dubois and Rezzonico, 2012, 2018).

Based on our current fragmentary knowledge, the following two chemotypes of Z. marina can be proposed:

A) Chemotype 1/ Baltic Sea: Luteolin 7,3 ′ - O -disulfate/diosmetin 7- O - sulfate type. This chemotype is characterized by a dominance of luteolin 7,3 ′ - O - disulfate 10 (24.7–58.7% of the total flavonoid pool) and diosmetin 7- O -sulfate 15 (17.4–41.3%). Moreover , this chemotype contains relatively low amounts of diosmetin 7,3 ′ - O - disulfate 17 (8.5–15.2%) and luteolin 7- O -sulfate 9 (4.1–15.1%), and very low amounts of apigenin 1 (1.3–7.2%) and luteolin 5 (1.5–7.5%). In contrast, luteolin 7- O - glucoside 7 reported from other parts of the range of Z. marina is absent or only occurring in trace amounts ( Enerstvedt et al., 2017). This chemotype was so far found in the Baltic Sea near Kiel / Germany.

B) Chemotype 2/ North Sea : Diosmetin 7- O -sulfate/luteolin 7,3 ′ - O - disulfate type. This chemotype is characterized by significant amounts of both luteolin 7,3 ′ - O -disulfate 10 (18.4–35.6%) and diosmetin 7- O - sulfate 15 (20.1–37.1%), with diosmetin 7- O -sulfate 15 being the dominant compound. Moreover, this chemotype contains more or less equal amounts of chrysoeriol 7- O -sulfate 16 (8.7–16.1%) and luteolin 7- O -sulfate 9 (5.4–15.0%), and relatively low amounts of luteolin 5 (4.1–6.7%) and diosmetin 7,3 ′ - O -disulfate 17 (3.7–8.1%). Additionally, the chemotype is characterized by the presence of apigenin 7- O -(6 ′′ - malonyl)-glucoside 4 and luteolin 7- O -(6 ′′ -malonyl)-glucoside 8. Z. marina collected in Espegrend coast, Norway belongs to this chemotype (data calculated from Enerstvedt et al., 2017).

Conclusively, we demonstrated that also in Z. marina different chemotypes seem to exist and that there is pronounced seasonal variation at least in the Baltic chemotype. In the future characterization of further chemotypes, great care should thus be taken not to confound seasonal (and other ecologic) effects with differences in chemotype. Based on our observations, early summer seems to be the optimal season for comparative investigations of phenol profiles in Z. marina .

In a related recent study, Dybsland et al. (2021) analyzed phenolic compounds in Z. marina from four locations in the Norwegian Sea area; the amounts of the detected compounds varied along environmental gradients. In particular, higher flavonoid contents were observed in the periphery of a seagrass meadow with a strong negative correlation of flavonoid contents with increasing depth of the sampling site ( Dybsland et al., 2021).

2.5. Bioactivity of 7 ʹʹ, 8 ʹʹ -didehydrosalvianolic acid B

7 ʹʹ,8 ʹʹ -Didehydrosalvianolic acid B is a new substance and it was therefore evaluated for cytotoxicity against three human cancer cell lines and against normal human fibroblasts. This compound showed no cytotoxicity against any of the tested cell lines after 72 h of treatment: breast adenocarcinoma, cervical carcinoma, malignant melanoma, and skin fibroblasts (data not shown). In addition, we also tested whether compound 21 may have any other biological activities, such as antiinflammatory activity, angiogenesis or ATPase inhibitory activity. We found that 7 ʹʹ,8 ʹʹ -didehydrosalvianolic acid B did not influence angiogenesis or inflammation. We also measured activity of ATPase after incubation with 21. Compound 21 inhibited the activity of this enzyme in the micro-molar range in a dose-dependent manner ( Fig. 7 ). Thus, our comprehensive biological screening revealed that 7 ′′,8 ′′ -didehydrosalvianolic acid B is able to specifically inhibit the activity of the isolated enzyme Na+ /K+ -ATPase, the inhibition of which could find practical use in the development of new drugs for prevention of cardiac hypertrophy and heart failure.

3. Conclusions

Z. marina from the Kiel Bay/Baltic Sea contained four phenolic acids and eight flavonoids with a prevalence of sulfated flavonoids. 7 ′′,8 ′′ - Didehydrosalvianolic acid B an undescribed tetrameric phenylpropanoid, was isolated and a Na + /K + -ATPase activity in the micro-molar range was proven. Detected variations in phenolic levels may reflect defensive strategies, adaptions to current environmental conditions, and/or protective factors against fouling. Regarding the existence of two Z. marina chemotypes in the German Baltic Sea (luteolin 7,3 ′ - O - disulfate/diosmetin 7- O -sulfate type) and the Norwegian North Sea (diosmetin 7- O -sulfate/luteolin 7,3 ′ - O -disulfate type), further studies are needed to elucidate a) whether more distinct chemotypes might exist, b) which are the areas of the chemotypes found in the current study, and c) whether these chemotypes are well separated from each other or connected by a clinal geographic gradient of intermediate forms. Comparing the amounts of phenolics found in Z. marina from the Norwegian coast ( Enerstvedt et al., 2017) and the German coast, a significantly higher concentration of natural products in the Norwegian populations of Z. marina was observed. Our preliminary results are not sufficient to unravel the reason(s) for these differences; these might be linked to climatic, other ecologic, and/or genetic factors. Understanding the underlined causes of the geographic and seasonal variation of Z. marina sulfated flavonoid chemistry and its possible link with ecological factors appears crucial to elucidating the ecologic roles of Zostera phenolics.

Figures

Fig. 7. Relative ATPase activity treated with 21 in micromolar range.