Bioactive natural products from marine algae and soft corals

Abstract

Natural products drug discovery from marine sources has received a remarkable attention in recent years given their potential biological activities. Second to none, natural products from marine origin exert a fascinating functionality owing to their wide range of structural diversity. Based on the literature, marine organisms including sponges, soft corals, cyanobacteria, microalgae, macroalgae, bryozoans, cnidaria, platyhelminthes, hoplonomertea, polychaetes, and hemichordate have extensively been studied for identifying numerous bioactive natural products those which are the secondary metabolites of these organisms. Some extensive reviews discuss the significance of marine natural products as bioactive agents [1, 2]. However, given the vast biological diversity of marine organisms widespread throughout the globe in different environmental habitats facing different physical and chemical environmental conditions, marine natural products remain under-explored. With recent developments which enable the exploration of marine habitats, the world has gone one foot forward in exploring fascinating marine organisms. Current series of studies merely represent a hay in a haystack contributing to the vast knowledge base of natural product drug discovery. The source of these studies were seaweeds harvested from the coast of Sri Lanka and soft corals harvested from ocean bordering Jeju island of the Republic of Korea. Following a general methodology of natural product drug discovery, extraction methods were designed to obtain targetted compound categories. Regarding seaweeds, the studies were focused upon the purification of bioactive polysaccharides and polyphenolic compounds. In the case of soft corals, the objectives were set to purify bioactive terpenoids and their derivatives. All selected fractions and purified compounds indicated potential functionality towards tested bioactivities unraveling their potential to be used in manufacturing a wide variety of consumer products. The studies incorporate the use of different chromatographic purification techniques, spectroscopic techniques, computational methods of molecular geometry optimization, spectroscopic analysis, and molecular docking and a variety of biological and chemical assays based on colorimetry, spectroscopy, western blot analysis and in vivo zebrafish model. The content of the thesis is divided into four parts to provide the reader a clear understanding of the workflow of current studies, which spread their branches to a variety of different areas. The first part of this thesis is devoted for the extraction, screening of bioactivities and purification of natural products from Sri Lankan marine algae. This section provides a general introduction to the importance of exploring marine natural products of seaweed featuring green, brown, red and microalgae and their significance in facilitating physiological wellbeing of man. In the first study crude polysaccharides were obtained from eleven different seaweed species by hot water extraction and ethanol precipitation. Among the investigated algae, the brown algae Chnoospora minima were found to be having better alkyl and DPPH radical scavenging activities and better intracellular ROS scavenging potential against both H_(2)O_(2) and AAPH induced ROS levels in "Chang" cells. FTIR analysis and monosaccharide composition of these crude polysaccharides were used to confirm its structural characteristics. During the interpretation of FTIR, a novel approach that combines the use of computational quantum chemical calculations in combination with predesignated structural information was followed. The calculations were done using density functional theory (DFT) calculations at RB3LYP/6-31G(d,p) level. Finally, the major constituent of the crude polysaccharides of Chnoospora minima was identified as fucoidan. In the next study, the green extraction approach that combines the use of enzymes to obtain a better yield of bioactive constituents were utilized in obtaining the water-based algae extracts. Ten different commercial food grade enzymes consisting of 5 carbohydrases (Viscozyme, Celluclast, AMG, Termamyl, and Ultraflo) and five proteases (Protamex, Kojizyme, Flavozyme, Alcalase, and Neutrase) were utilized in obtaining the extracts provided their optimum extraction conditions. Celluclast extracts gave the highest yield for all the algae samples with higher amounts of polyphenolic contents. The antioxidant and anti-inflammatory properties of the Celluclast extracts of Sargassum polycystum and Chnoospora minima were identified superior compared to other extracts using both in-vivo and in-vitro analysis. As for the third study, the olysaccharides rich in fucoidans were precipitated out from the Celluclast extract of C. minima using ethanol and further purification was done via a DEAE-Sepharose fast flow column. Four fractions were collected from the column purification. One of the purified fraction demonstrated excellent anti-inflammatory effects compared to other three fractions in-vivo and in-vitro. The selected fraction was found to be a fucoidan with a higher fucose content and degree of sulfation via FTIR characterization and monosaccharide analysis. The results further demonstrated the variation of anti-inflammatory properties with the degree of sulfation of the polysaccharide backbone. In the next study, we optimized a method to obtain fucoidan-rich polysaccharides from S. polycystum and C. minima following depigmentation, phenol polymerization, enzyme-assisted extraction, removal of alginate impurities by CaCI_(2) addition and ethanol precipitation. The obtained polysaccharides demonstrated the characteristics of fucoidan evidenced via both FTIR analysis and monosaccharide composition analysis. Further, they demonstrated superior antioxidant properties for DPPH and Alkyl radical scavenging activities. Also, the fucoidan-rich polysaccharides demonstrated desirable bioactive properties such as anti-inflammatory, tyrosinase inhibition, skin whitening, elastase and collagenase inhibition abilities making them much desirable natural ingredients to be used in the formulation of cosmeceutical products. Apart from studying about the fascinating bioactive polysaccharides of algae, 70% ethanol extracts of some selected algae were evaluated for their bioactive properties. The algae (Ahnfeltiopsis pygmaea, Gracilaria corticata var. ramalinoides, Chnoospora minima, and Caulerpa racemosa) extracts obtained in such manner was found to composed of a higher level of polyphenolic content when compared to their water based extracts described previously. Among the extracts, C. minima, and C. racemose demonstrated superior antioxidant and antiinflammatory effects. Also, the C. minima indicated potential anti-cancer activities against HL-60 cell line. The crude of selected C. minima and C. racemose extracts were dissolved in water and fractionated between inert organic solvents in order of increasing their polarity following hexane, Chloroform, and ethyl acetate. The ethyl acetate fraction of C. minima and C. racemose demonstrated strong antioxidant and anti-inflammatory properties whereas the hexane fraction indicated anti-inflammatory activity. Chloroform fractions of C. racemose indicated potential anti-cancer properties against HL-60 (human leukemia) and MCF-7 (human breast cancer) cells. Further purification of the hexane fraction guided by bioassays (anti-inflammatory activity) resulted in the isolation of Squalene, which was confirmed based on its molecular weight, and NMR data. The purified compound indicated a desirable anti-inflammatory effect on LPS-induced RAW 264.7 murine macrophages by the reduction in NO production and down-regulation of pro-inflammatory cytokines and inflammatory regulators. Further, the antiinflammatory implications of the isolated compound were confirmed in the LPS-induced in vivo zebrafish embryo model. Investigation of soft corals harvested from Jeju describes the next part of the study. Soft corals are species belongs to the order of Alcyonacea, which defines corals that do not produce a calcium carbonate skeleton. They primary inhabit nutrition rich tropical or subtropical waters favoring habitats with less light and warm seawaters. Based on the estimates from 2010 to 2011, 22% of the total new marine natural products have been discovered from soft corals [3]. Natural products from soft corals are considered as a primary source of a new array of therapeutics. Surprisingly, most of the natural products identified from soft corals have reported demonstrating a spectrum of biological activities including antitumor, antiviral, antifouling and anti-inflammatory effects [4]. Given their evolutionary perspectives, these compounds many of which are toxic to other organisms assist the soft corals in protecting themselves from predators [5]. The soft corals belong to the genus Dendronephthya that was central to current studies encompasses nearly 248 species and are highly prolific and widely distributed throughout the Indian Ocean, the Red Sea, Pacific Ocean and Southeast Asia [6]. Especially the sterols isolated from soft corals have shown promising bio functionalities. A review about marine sterols published in 1993 report the discoveries been done on more than 200 different types of mono hydroxy sterols from marine organisms [7]. Discovery of these various metabolites will shape the future of natural product drug development with a broad perspective of counteracting a broad range of disease conditions. Ten different soft coral species including Dendronephthya gigantea, Dendronephthya spinulosa, Dendronephthya puetteri, Dendronephthya castanea, Dendronephthya aurea, Dendronephthya suensoni, Scleronephthya gracillimum, Chromonephthea hirotai and two unknown Dendronephthya species designated Dendronephthya sp1 and Dendronephthya sp2 were extracted using 70% ethanol for the initial evaluation of its bioactive properties. The 70% ethanol extracts of Dendronephthya gigantea, Dendronephthya aurea, Dendronephthya puetteri, Chromonephthea hirotai, and Scleronephthya gracillimum indicated promising antiinflammatory effects against LPS-induced NO production, down-regulation of proinflammatory cytokines and inflammatory mediators. Among the screened soft corals Dendronephthya gigantea was selected for further studies to purify bioactive principals responsible for the observed anti-inflammatory and anti-cancer effects. The solvent/solvent fractionation of the 70% ethanol extract obtained by 6.00 Kg of the Dendronephthya gigantea dry powder indicated the accumulation of the anti-inflammatory and anti-cancer effects in the hexane fraction. Following the bioassays, the hexane fraction was further purified by two consecutive silica open columns to obtain a subfraction with highest potential bioactive properties. The selected fraction indicated a remarkable anti-inflammatory activity towards LPS-induced RAW 264.7 macrophages evidenced via the reduced NO production, downregulation of pro-inflammatory cytokines and inflammatory mediators. Effects were also displayed in LPS-induced in vivo zebrafish model. The active principals in the purified fraction were identified as a mixture of eight 3β-hydroxy-Δ5-steroidal congeners via GC-MS/MS analysis. Following the impressive biological functionality of the purified fraction, attempts were taken to isolate the individual compounds in the sterol mixture. The separation of sterols is a tricky task that needs both accuracy and precision. For the further separation, a long silica column was used with gradually increasing amounts of ethyl acetate in hexane mixture. Column eluates were collected into 130 test tubes and grouped into five fractions. Based on its TLC analysis patterns, the selected test tubes were mixed with each other and further developed on a preparative TLC plate. The separated bands were collected from the TLC plates and analyzed via GC-MS/MS and NMR. The isolated sterols are remaining to be screened for some possible bioactivities including anti-inflammatory, anti-cancer, and anti-diabetic. Hence, the progressions in marine bioresource technology would provide a new scope and a platform for the developments in natural product chemistry, functional food, cosmeceutical, and pharmaceutical industry.