Angiotensin I-Converting Enzyme (ACE) Inhibition and Molecular Docking Study of Meroterpenoids Isolated from Brown Alga, Sargassum macrocarpum

Angiotensin I-converting enzyme (ACE) is an important blood pressure regulator. In this study, we aimed to investigate the ACE-inhibitory effects of meroterpenoids isolated from the brown alga, Sargassum macrocarpum, and the molecular mechanisms underlying ACE inhibition. Four fractions of S. macrocarpum were prepared using hexane, chloroform, ethyl acetate, and water as solvents and analyzed for their potential ACE-inhibitory effects. The chloroform fraction showed the strongest ACE-inhibitory effect, with an IC50 value of 0.18 mg/mL. Three meroterpenoids, sargachromenol, 7-methyl sargachromenol, and sargaquinoic acid, were isolated from the chloroform fraction. Meroterpenoids isolated from S. macrocarpum had IC50 values of 0.44, 0.37, and 0.14 mM. The molecular docking study revealed that the ACE-inhibitory effect of the isolated meroterpenoids was mainly attributed to Zn-ion, hydrogen bonds, pi-anion, and pi–alkyl interactions between the meroterpenoids and ACE. These results suggest that S. macrocarpum could be a potential raw material for manufacturing antihypertensive nutraceutical ingredients.


Introduction
Hypertension is a chronic health problem related to cardiovascular diseases, including myocardial infarction, arteriosclerosis, and stroke, and is affected by various factors, such as salt intake, smoking, stress, and obesity [1]. Among the processes associated with hypertension, angiotensin I-converting enzyme (ACE), which is involved in the kallikrein-kinin system (KKS) and renin-angiotensin system (RAS), plays a pivotal role in the development of hypertension [2][3][4]. ACE is a zinc metalloproteinase [5]. It is found in the vascular, heart, lung, and brain tissues [6]. In the KKS, ACE plays an important role in degrading the potent vasodilator bradykinin, while in the RAS, ACE catalyzes the conversion of the potent vasoconstrictor octapeptide angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) from the inactive decapeptide angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) [7]. Controlling blood pressure control by inhibiting ACE activity is regarded as a preventive and therapeutic strategy for hypertension. The synthetic ACE inhibitors, including captopril, enalapril, ramipril, and benazepril, are extensively used for the treatment of essential hypertension in humans [6, 8,9]. However, these synthetic inhibitors have side effects, such as taste disturbances, skin rashes, and coughing [10,11]. Therefore, studies are focusing on using natural resources as potential ACE-inhibitory agents against hypertension.

Isolation and Identification of the Compounds
Next, the chloroform fraction was subjected to silica gel electrophoresis and MPLC. Finally, the compounds in this fraction were isolated using preparative HPLC and identified as meroterpenoids based on a comparison between the NMR spectroscopic data and previous literature [29,30]. Sargachromenol
S. macrocarpum. Moreover, meroterpenoids are promising for the discovery of potential candidates for the future industrial production of functional foods.

Materials
S. macrocarpum was collected from Jeju Island, South Korea. The alga was washed three times with tap water to remove salt and epiphytes attached to the surface, followed by careful rinsing with fresh water. The algae were stored in a medical refrigerator at −80 °C. Frozen algae were lyophilized and homogenized using a grinder prior to extraction. The ACE assay kit (ACE kit-WST) was obtained from Dojindo Laboratories (Kumamoto, Japan).

Extraction and Isolation of Compounds
The dried S. macrocarpum powder was extracted with 80% aqueous methanol at room temperature, and the liquid layer was obtained via filtration. The filtrate was evaporated to obtain the methanol extract, which was suspended in distilled water and partitioned with hexane, chloroform, ethyl acetate, and butanol. The chloroform fraction exhibited a stronger ACE-inhibitory effect than the other fractions. Thus, the compounds were extracted from the chloroform fraction using a silica gel column and medium-pressure liquid chromatography (Selekt MPLC system, biotase, Uppsala, Sweden). Finally, the compounds were purified using preparative high-performance liquid chromatography (Nextra prep-HPLC, Shimadzu, Kyoto, Japan), and the structures of the compounds were identified by comparing the nuclear magnetic resonance (Varian VNMRS 500 MHz FT-NMR spectrometer, Varian, Pala Alto, CA, USA) spectral data and high-resolution electrospray ionization mass spectrometry (SCIEX X500R Q-TOF LC-MS/MS spectrometer, SCIEX, Framingham, MA, USA) analysis results with those in the existing literature.  site contributed to the stabilization of the docking complex, and it was confirmed that sargaquioic acid contributed the most to stabilization. These findings provide a partial molecular explanation for the ACE-inhibitory properties of meroterpenoids isolated from S. macrocarpum. Moreover, meroterpenoids are promising for the discovery of potential candidates for the future industrial production of functional foods. site contributed to the stabilization of the docking complex, and it was confirmed that sargaquioic acid contributed the most to stabilization. These findings provide a partial molecular explanation for the ACE-inhibitory properties of meroterpenoids isolated from S. macrocarpum. Moreover, meroterpenoids are promising for the discovery of potential candidates for the future industrial production of functional foods. site contributed to the stabilization of the docking complex, and it was confirmed that sargaquioic acid contributed the most to stabilization. These findings provide a partial molecular explanation for the ACE-inhibitory properties of meroterpenoids isolated from S. macrocarpum. Moreover, meroterpenoids are promising for the discovery of potential candidates for the future industrial production of functional foods.

R-sargachromenol
S-sargachromenol R-7-methyl sargachromenol S-7-methyl sargachromenol sargaquinoic acid site contributed to the stabilization of the docking complex, and it was confirmed that sargaquioic acid contributed the most to stabilization. These findings provide a partial molecular explanation for the ACE-inhibitory properties of meroterpenoids isolated from S. macrocarpum. Moreover, meroterpenoids are promising for the discovery of potential candidates for the future industrial production of functional foods.

R-sargachromenol
S-sargachromenol R-7-methyl sargachromenol S-7-methyl sargachromenol sargaquinoic acid site contributed to the stabilization of the docking complex, and it was confirmed that sargaquioic acid contributed the most to stabilization. These findings provide a partial molecular explanation for the ACE-inhibitory properties of meroterpenoids isolated from S. macrocarpum. Moreover, meroterpenoids are promising for the discovery of potential candidates for the future industrial production of functional foods.   In conclusion, among the various organic solvent fractions, the chloroform fraction exhibited the highest ACE inhibitory effect. Three meroterpenoids, sargachromenol, 7-methyl sargachromenol, and sargaquinoic acid, having ACE-inhibitory effects were isolated, with IC 50 values 0.44, 0.37, and 0.14 mM, respectively. Among the three meropterpenoids, sargachromenol and 7-methyl-sargachromenol do exist as Rand S-isomers, and molecular docking studies of the respective other isomers were performed. These other isomers showed the similar docking poses and binding energies with those of isomers. Molecular docking studies revealed that these meroterpenoids in the ACE active site contributed to the stabilization of the docking complex, and it was confirmed that sargaquioic acid contributed the most to stabilization. These findings provide a partial molecular explanation for the ACE-inhibitory properties of meroterpenoids isolated from S. macrocarpum. Moreover, meroterpenoids are promising for the discovery of potential candidates for the future industrial production of functional foods.

Materials
S. macrocarpum was collected from Jeju Island, South Korea. The alga was washed three times with tap water to remove salt and epiphytes attached to the surface, followed by careful rinsing with fresh water. The algae were stored in a medical refrigerator at −80 • C. Frozen algae were lyophilized and homogenized using a grinder prior to extraction. The ACE assay kit (ACE kit-WST) was obtained from Dojindo Laboratories (Kumamoto, Japan).

Extraction and Isolation of Compounds
The dried S. macrocarpum powder was extracted with 80% aqueous methanol at room temperature, and the liquid layer was obtained via filtration. The filtrate was evaporated to obtain the methanol extract, which was suspended in distilled water and partitioned with hexane, chloroform, ethyl acetate, and butanol. The chloroform fraction exhibited a stronger ACE-inhibitory effect than the other fractions. Thus, the compounds were extracted from the chloroform fraction using a silica gel column and medium-pressure liquid chromatography (Selekt MPLC system, biotase, Uppsala, Sweden). Finally, the compounds were purified using preparative high-performance liquid chromatography (Nextra prep-HPLC, Shimadzu, Kyoto, Japan), and the structures of the compounds were identified by comparing the nuclear magnetic resonance (Varian VNMRS 500 MHz FT-NMR spectrometer, Varian, Pala Alto, CA, USA) spectral data and high-resolution electrospray ionization mass spectrometry (SCIEX X500R Q-TOF LC-MS/MS spectrometer, SCIEX, Framingham, MA, USA) analysis results with those in the existing literature.

Determining the ACE Inhibitory Activity
The ACE-inhibitory activities of the extract, fractions, and compounds were determined using an ACE kit-WST (Dojindo Laboratories). The assay was performed according to the manufacturer's instructions. The absorbance was measured at 450 nm using a microplate reader (Model 550, Bio-Rad, Hercules, CA, USA). IC 50 value was defined as the concentration of extract and fractions (mg/mL), and compounds (µM) required for 50% reduction of the ACE activity.

Three-Dimensional (3D) Structure of the Proteins and Compounds
Molecular docking was performed using CDOCKER and Calculate Binding Energies tools in Discovery Studio 2022 (Biovia, San Diego, CA, USA) to assess the binding positions of meroterpenoids within the active site of ACE. A docking mechanism based on CHARMm [41] was used to execute the CDOCKER docking protocol. The docking of marine aldehyde derivatives to the ACE was performed as follows: (1) a two-dimensional (2D) structure was converted to a 3D structure, (2) proteins were prepared, and the binding sites were defined, and (3) the compounds were docked [42]. The ACE-binding pocket was defined as the area from the center of the active site up to a radius of 11.2 Å. The binding site and ligand were allowed to move freely during the docking. Water molecules were removed from the protein in the flexible docking process because the fixed water molecules might alter the generation of the ligand-receptor complex. After the removal of water molecules, hydrogen atoms were attached to the protein. Ligand binding affinity was assessed for all complexes by applying the CHARMm force field to the interaction energy. Based on CDOCKER's interaction energy, the distinct conformational positions for each molecule were identified and examined. The binding energies of the small-molecule protein complexes were calculated using the Calculated Binding Energies tool and used to select candidate compounds. The docking positions of the selected compounds to ACE are expressed as 2D and 3D crystal structures.

Statistical Analyses
Data are presented as mean ± standard error. Statistical comparisons of the mean values were performed using analysis of variance, followed by Duncan's multiple-range test using the SPSS software (Version 21). A p < 0.05 was considered statistically significant.