For the potential isolation of ChEs inhibitors from natural marine products, the ethanolic extracts of 27 Korean seaweeds were evaluated using a bioassay-guided fractionation strategy. The ChEs inhibitory properties of the ethanolic extracts, observed at concentrations of 100.0 μg/mL, are listed in Table 1. Of the seaweeds tested, the ethanolic extracts of E. stolonifera and E. cava, belonging to the Ecklonia species, exhibited detectable ChEs inhibitory activities against both AChE and BChE, with inhibition of 45.97 ± 3.10 and 30.90 ± 4.20%, and 21.28 ± 2.21 and 13.14 ± 2.24%, respectively. In particular, E. stolonifera showed more potent ChEs inhibitory activity than E. cava, probably due to the compositional difference between their active components. Although Sargassum sagamianum has previously been reported to exhibit BChE inhibitory activity,14 our studies detected no such effects with the Sargassum sp. tested, i.e. S. fulvellum, S. horneri and S. thunbergii, which may be attributable to differences in the constituents between the species. Therefore, the ethanolic extract of E. stolonifera was chosen for further studies.
In the present ongoing study for the identification of active components, the ethanolic extract of E. stolonifera was successively partitioned with n-hexane, CH2Cl2, EtOAc and n-BuOH, yielding the respective fractions, with the inherent inhibitory activities of each then assessed. As shown in Table 2, the CH2Cl2 (71.52 ± 5.46 μg/mL), EtOAc (26.46 ± 4.76 μg/mL) and n-BuOH (82.15 ± 3.23 μg/mL) soluble fractions exhibited significant AChE inhibitory activities. The EtOAc soluble fraction of the ethanolic extract from E. stolonifera had noticeable inhibitory activity towards AChE. Conversely, the n-hexane (68.40 ± 4.03 μg/mL) and CH2Cl2 (71.10 ± 3.54 μg/mL) fractions had profound inhibitory activity against BChE. However, the H2O fraction showed no detectable activity towards the ChEs, suggesting the active ingredients of the EtOH extract of E. stolonifera were included in the moderately polar EtOAc fraction and non-polar n-hexane fraction for AChE and BChE, respectively. The comparative inhibitory activities toward ChEs of the fractions derived from the EtOH extract of E. stolonifera can be explained by the presence of different components. The activities of both the EtOAc and n-hexane fractions indicated that the inhibitory principles were phlorotannins and sterols, as they are known to widely exist in Ecklonia sp. and have polar solubilities in EtOAc and n-hexane, respectively.47,54 Therefore, the most active EtOAc and n-hexane soluble fractions were subjected to further chemical analysis, with repeated column chromatography of the active n-hexane and EtOAc fractions leading to the isolation of two sterols, fucosterol (1) and 24-hydroperoxy 24-vinylcholesterol (2), from the n-hexane fraction, and eight phlorotannins, phloroglucinol (3), eckstolonol (4), eckol (5), phlorofucofuroeckol-A (6), dieckol (7), triphlorethol-A (8), 2-phloroeckol (9) and 7-phloroeckol (10), from the EtOAc fraction. Of these compounds, 24-hydroperoxy 24-vinylcholesterol (2), 2-phloroeckol (9) and 7-phlorockol (10) were isolated from E. stolonifera for the first time in this study; their structures were also verified by comparison with published data.32,55,56 The structures of the isolated compounds are presented in Figure 1, with their ChEs inhibitory effects shown in Table 3. Of the isolated compounds, compounds 4–7, 9 and 10 exhibited significant AChE inhibitory activities, with IC50 values of 42.66 ± 8.48, 20.56 ± 5.61, 4.89 ± 2.28, 17.11 ± 3.24, 38.13 ± 4.95 and 21.11 ± 4.16 μM, respectively. In the AChE assay, compound 2 showed marginal inhibitory activity, with an IC50 value of 389.10 ± 2.29 μM. Conversely, compounds 1, 2, 4 and 6 exhibited moderate inhibitory activities against BChE, with IC50 values of 421.72 ± 1.43, 176.46 ± 2.51, 230.27 ± 3.52 and 136.71 ± 3.32 μM, respectively. However, compounds 3 and 8 showed no activity toward either AChE or BChE.