Characterization of bioactive components from Ishige okamurae and their biological activity

Abstract

Seaweeds are one of the crucial marine living renewable resources as they are able to produce a great variety of secondary metabolites characterized by a broad spectrum of biological activities. Ishige okamurae (I. okamurae) is a member of the family Ishige as an edible brown alga, have attracted attention as various natural compounds with their biological activities. In this study, new separation method was suggested to increase efficiency and improve the isolation performance using Pure C-850 FlashPrep system. In this study, Pure C-850 FlashPrep system was suggested to increase efficiency and improve the isolation of natural compounds from I. okamurae. A novel series of polyphenols, named as Ishophloroglucin; Ishophloroglucin A (IPA), Ishophloroglucin B (IPB), Ishophloroglucin C (IPC), and Ishophloroglucin D (IPD), and another two known compounds which is α/β-adenosine and Diphlorethohydroxycarmalol (DPHC) were isolated and identified. Their structure was elucidated by 1D- and 2D- Nuclear magnetic resonance (NMR), including 1H, 13C, HMBC, and COSY, as well as liquid chromatography-tandem mass spectrometry (LC-MS/MS). As folk medicine, I. okamurae with various biological effects grows abundantly on the shores of Japan, north of China, and Korea where it is consumed as a part of daily diet. Appropriate quality control operations shall be employed to ensure that food having uniformed nutrients is suitable for human consumption. This study was conducted to establish an HPLC analysis method for determination of marker compounds as part of materials standardization for development of health functional food materials from I. okamurae. DPHC was selected as a marker compound due to it is more stable compared to IPA on the thermal stability analysis. The method was validated by system suitability, specificity, linearity, limits of detection (LOD), limits of quantification (LOQ), precision, and accuracy. The method showed high linearity of the calibration curve with a coefficient of correlation (R2) of 0.9999, and LOD and LOQ was 0.144 μg/mL and 0.435 μg/mL, respectively. Relative standard deviation values from precision was less than 0.622 %. Recovery rates of DPHC was 106.35 - 107.82 % within 100 ± 20%. An optimized method for extraction of DPHC in I. okamurae was established through diverse extraction conditions, and the validation indicated that the method is accurate and sensitive for the determination of marker compounds in I. okamurae to develop a health functional food material. Polyphenols, a group of phloroglucinol (1,3,5-trihydroxybenzene), are the dominant polyphenolic secondary metabolites found in marine brown algae. IPA as a dominant polyphenol in I. okamurae, was investigated their anti-melanogenesis effects. Through the comparative molecular docking analysis of IPA to tyrosinase (3NM8), IPA was further investigated their inhibitory effect against tyrosinase activity and melanin formation that causes an increase anti-melanogenesis effect in both murine melanoma cells in-vitro model and zebrafish in-vivo model. The α/β-adenosine (1:1 ratio) was firstly identified from I. okamurae, which is known as a naturally occurring substance that relaxes and dilates blood vessels in angiogenesis. To characterize the vasodilation effect of α/β-adenosine in endothelial dysfunction of angiogenesis induced by Particulate matter (PM) exposure, commercial isomer β-adenosine was also investigated as well in human endothelial cell line EA.hy926 in-vitro model and transgenic zebrafish in-vivo model. In the last study, fucoidan is an interesting group of bioactive sulfated polysaccharides in brown seaweeds. The current study highlights the enrichment and extraction of fucoidan from I. okamurae through enzyme-assistant extraction using Celluclast enzyme. The structural characterization of fucoidan was performed using FTIR and NMR spectroscopy, their monosaccharide compositions was analyzed by HPAE-PAD, the molecular weight distribution was performed by agarose gel electrophoresis and the sulfate content was analyzed as well. The purified fucoidan and its anti-inflammatory activity was investigated in-vitro and in-vivo. In summary, this research suggested various natural compounds from I. okamurae isolated by a flash prepare system and their potential uses as marker compounds or pharmaceutical and nutraceutical agents. Therefore, the purpose of this research is to discuss the potential health benefits of I. okamurae to be used as a functional material in industries such as functional foods, nutraceuticals, and cosmeceuticals to utilize this precious natural resource for future generations.