Structural characteristics and biological activities of fucoidans extracted from selected Vietnamese brown seaweeds

MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ...*** BUI VAN NGUYEN STRUCTURAL CHARACTERISTICS AND BIOLOGICAL ACTIVITIES OF FUCOIDANS EXTRACTED FROM SELECTED VIETNAMESE BROWN SEAWEEDS Major: Chemistry of natural compounds Code: 9440117 SUMMARY OF CHEMISTRY DOCTORAL THESIS Ha Noi – 2018 This research has been done at Graduate University of Science and Technology – Vietnam Academy of Science and Technology Supervisor 1: Asc.Prof.Dr. Bui Minh Ly Supervisor 2: Asc.Prof.Dr. Nguyen Quyet Chien Reviewer 1: Reviewer 2: Reviewer 3: The dissertation will be defensed before the Evaluation Council of the doctoral dissertation at the Academy, meeting at the Academy of Science and Technology - Vietnam Academy of Science and Technology at ... hours ... ', date ... month 2018. The dissertation can be found at: - Library of the Graduate University of Science and Technology - National Library of Vietnam 1 INTRODUCTION Viet Nam is recognized internationally as one of the countries that has the world’s highest biodiversity with various types of forest, swamps, river and stream, sea, etc. Located in the center of the South East Asia, Viet Nam’s total coastline is about 3260 km long as the west border of the East Sea with the area of over 1,000,000 km2, it is one of the world’s most important seas having various and abundant algae. There are about 6000 identified species of seaweed in the world and they are divided into 3 main seaweed, divisions in terms of pigment: green algae (Chlorophytes), brown algae (Pheophytes) and red algae (Rhodophytes). Algae plays an important role in sea creature resources. They are being widely exploited, raised and used more and more by human in food and industry. According to research results, Viet Nam now explores nearly 1000 seaweed species, 143 of which are brown algae (Phaeophyta) that have large, long individual size as well as large number capacity. Therefore, brown algae are considered as a valuable raw material of the present and the future of agriculture, drug manufacturing industry, functional food and cosmetic. In the drug manufacturing industry, brown algae are used as the main raw material to extract compounds that have biological activity with large application capacity. Marine brown algae, representatives of the class Phaeophyceae, are known as a rich source of unique polysaccharides. Among them, alginic acids and alginates have found various technical applications, whereas sulfated polysaccharides (fucoidans) are intensively studied in view of their various promising biological activities, mainly anticoagulant and antitumor ones. Fucoidans are sulfate polysaccharides derived from marine brown seaweed containing mainly fucose and sulfate groups with other residues such as galactose, xylose, glucose, manose and uronic acids. Fucoidans were reported to possess various biological effects in vitro and in vivo such as anti-inflammatory, anticoagulant, antithrombotic, antiviral including anti-HIV, immunomodulatory, antioxidant and antitumor. Thanks to the variety of chemical structure and having many interesting biological activities, fucoidan has been strongly researched. Elucidation of fine chemical structure of fucoidans is complicated as fucoidan preparations are often mixtures of structurally different sulfated polysaccharides. These usually have branched non-regular structures and contain different monosaccharides and noncarbohydrate substituents (sulfate and acetate groups). Fucoidans from algae belonging to the family Sargassaceae have especially complex structures, but are attractive since these algae are readily available from natural populations. Fucoidan from brown algae belongs to Sargassum, Hormophysa and Turbinaria, has complicated chemical structure but is very appealing to activity as well as its application and available in the nature. Although many researches in order to identify fucoidan’s sophisticated structure are published, only few results find out the regularity of fucoidan’s structure such as the association among sugar residues bases, embranchment, the position of sulfate bases and other monosaccharide molecules. Up to now, most of the researches on biological activity have been carried out on crude fucoidan. Therefore, the relationship between structure and biological activity of fucoidan in reality, up to now, has not been cleared. To help the research on effect mechanism of fucoidan on biological cells and using fucoidan to prepare medicines, accurate identification of fucoidan’s chemical structure is the first decision and attracting many scientists around the world. Therefore, I have chosen the topic: “Structural characteristics and biological activities of fucoidans extracted from selected vietnamese brown seaweeds”. The research goals of the dissertation: The research separates, identifies structural characteristics and tests biological activity of fucoidan 2 from several brown algae growing up in Vietnamese Seas to serve for investigating Vietnamese natural sea compound resources and making clear the chemical nature of research objects. In order to achieve the above targets of the thesis, the research contents of the thesis include: Studying and screening some brown seaweed species to select the subjects for in depth research on the structure as well as biological activities of fucoidan. Making an in-depth study of the structure as well as the biological activity of fucoidan extracted from selected brown seaweed species. Investigating the relationship between structural characteristics and biological activity of selected fucoidan. Structure of the dissertation: The study consists of 175 typewritten pages with 31 tables and 64 figures. For instance, 3 pages of introduction, 45 pages of overview, 21 pages of research methods, 15 pages of experiment, 79 pages of result and discussions, 4 pages of conclusions and recommendations, 2 pages of publication list, and 12 pages of references. Chapter 1. OVERVIEW Seaweed, also known as macroalgae is an autotrophic lower plant by photosynthesis, in shape of thallus. The seaweed grows fast with the growing life span of no more than one year, rapid growth speed and creates massive biomass. The total number of seaweed species reported in the world mainly belong to three main divisions, 900 species are of green seaweed (Chlorophyta), 1500 species are of brown seaweed (Phaeophyta) and 4000 species are of red seaweed (Rhodophyta), and there has been many new species being discovered in studies added to the total number of the species of seaweed distributed throughout the world. Fucoidans are sulfated polysaccharides derived from brown seaweed which was isolated from brown seaweed firstly by Kylin in 1913. According to its nomenclature namely carbohydrate, it is because the polysaccharides are formed by fucose and sulfate is named fucan sulfate. Sulfated polysaccharides originated from brown seaweed and animals is actually present in echinoderms, specially urticaria and sea cucumbers. In contrast, the structure of sulfated polysaccharides derived from brown seaweed is much more complex in its composition. In addition to fucose and sulfate, they can also contain other monosaccharides such as galactose, xylose, manose, glucuronic acid, etc as well as can be partially acetylated. The specific chemical structure of these complex biopolymers is unknown in many cases. Sulfated polysaccharide (Fucoidan) is a compound which attracts a lot of interest due to its diverse and unique biological activities. These include anticoagulant, antithrombotic, antiviral, antiangiogenic, anti-inflammatory, anti-tumor, anticomplementary, modulating the immune system, contraception. Thus, fucoidan has become a potential source of functional foods, nutritious foods, medicinal products, etc and the number of studies on fucoidan has increased strongly in the last 10 years. After having studied the overview situation of domestic and abroad studies, we draw the following conclusions: Studies on the content, chemical composition and structural characteristics of fucoidan extracted and isolated from brown seaweeds of Sargassum feldmannii, Sargassum duplicatum, Sargassum denticapum and Sargassum binderi, these have not been fully and systematically researched. For studies on the structure and activity of fucoidan from two brown seaweed species being studied in the study, namely Sargassum aquifolium and Turbinaria decurrens, these has not been studied in domestic and abroad studies. The chemical structure of fucoidan from 6 brown seaweed species including Sargassum polycystum (Fsp), 3 Sargassum mcclurei (Fsm), Sargassum oligocystum (Fso), Sargassum denticarpum (Fsd), Sargassum swatzii (Fsw) and Tubinaria ornata (Fto) has been studied in previous studies, however, none of them studies on the relationship between branching structure of fucoidan with its cytotoxic activity. Therefore, we will conduct the study on the relationship between shape and size with the biological activity of branched fucoidan from 6 above-mentioned species. Chapter 2. SUBJECT AND RESEARCH METHODS 2.1. Subject of the study The subjects of research of the thesis are fucoidans isolated from some species of brown seaweed. Brown seaweeds are collected at the seas in Vietnam. The specific subjects of the thesis are as follows: Fucoidan isolated from brown seaweeds Sargassum aquifolium (FSA) and Turbinaria decurrens (FTD), these fucoidans are used to study the chemical structure and biological activity in the thesis. Two brown seaweed species are main subjects of the thesis. Fucoidan from 6 brown seaweed species: Sargassum polycystum (Fsp), Sargassum mcclurei (Fsm), Sargassum oligocystum (Fso), Sargassum denticarpum (Fsd), Sargassum swatzii (Fsw) and Tubinaria ornata (Fto), which have been studied their structure, which is used to study the relationship between structure and biological activity. Fucoidan from brown seaweed species Sargassum feldmannii, Sargassum duplicatum, Sargassum denticapum and Sargassum binderi which is the study subject of chemical composition and structural characteristics of fucoidan. 2.2. Research methods - Extraction and Purification of Fucoidan: The method of Bilan et al - Chemical Analysis: monosaccharides, sulfate content and uronic acid. - Anion-Exchange Chromatography. - Structural determination of fucoidan: Gel Permeation Chromatography (GPC), IR spectra, ESI-MS spectra, Small Angle X-ray Scattering (SAXS), NMR spectra: 1H-NMR, 13C-NMR, COSY, HSQC, HMBC, - Chemical methosd: Desulfation, methylation analysis. - Biological activity assay. Chapter 3. EXPERIMENT 3.1. Seaweed collection and identification Brown seaweed species were harvested from Nha Trang bay, Khanh Hoa province, Viet Nam. The samples were collected in May 2014 and identified by Dr Le Nhu Hau (Nha Trang Institute of Technology Research and Application). A voucher specimen named are deposited in Nha Trang Institute of Technology Research and Application. The sample was washed in seawater to remove mud, sand and other substances and then air-dried at room temperature and milled to fine powder. The samples used for extraction and purification of fucoidan. 4 3.2. Extraction and purification of fucoidan from brown seaweed species Figure 3.3. Processing for the extraction and purification of fucoidans from Sargassum aquifolium Figure 3.4. Processing for the extraction and purification of fucoidans from Turbinaria decurrens 5 Figure 3.5. Processing for the extraction and purification of fucoidan from S.polycystum (Fsp), S.mcclurei (Fsm), S.oligocystum (Fso), S.denticarpum (Fsd), S.swatzii (Fsw) and T.ornata (Fto) Figure 3.6. Processing for the extraction and purification of fucoidans (Patent WO 2005/014657) 6 3.3. Analyze the chemical components and structural determination of fucoidan 3.3.1. Total carbohydrate by phenol-sulfuric acid 3.3.2. Monosaccharides compositions were eclucidated by the method of Bilan et al 3.3.3. Sulfate content was determined by following the method of Dodgson et al 3.3.4. Sulfation method 3.3.5. Uronic acid content was determined by following the method of Bitter et al 3.3.6. Methylation analysis 3.3.7. Preparation for oligosaccharide 3.3.8. Gel Permeation Chromatography (GPC): The weight average molecular mass and the number average molecular mass were elucidated on a HPLC Agilent 1100. 3.3.9. IR Spectra: IR spectra was recorded on a FT-IR Brucker 3.3.10. NMR Spectra (1H-NMR, 13C-NMR, COSY, HSQC, HMBC, ): NMR spectra were recorded on Bruker Avance III 500MHz and Bruker Avance 600MHz. 3.3.11. ESI-MS/MS Spectra: ESI-MS spectra were recorded on Xevo TQ MS, Water-USA. 3.3.12. Small Angle X-ray Scattering (SAXS): SAXS experiments were performed at BL10C, Photon Factory, Tsukuba, Japan. 3.4. Biological activity assay: Measurement of cytotoxicity, measurement of antitumor activity, measurement of anticoagilant activity Chapter 4. RESULTS AND DISCUSSIONS 4.1. The study chooses the fucoidan extraction method We have applied the extraction process of fucoidan from the method of Bilan et al to study the structure and biological activity of fucoidans. 4.2. Content of fucoidan and of some water soluble polysaccharides of 06 brown seaweed species grown in Nha Trang beach, the chemical composition of the fucoidans obtained. The content of fucoidan were determined by way of direct isolation according to the method of Bilan et al. The content of laminaran and alginate were determined by conventional extraction in the procedure of fucoidan isolation. The analyzing results show that: Content of fucose occupied significantly from 9.2 to 62.9% in all fucoidan samples, in which the highest fucoidan content was fucoidan extracted from Turbinaria decurrens (62.9%) and the lowest was fucoidan extracted from Sargassum aquifolium (9.2% ). Galactose content of fucoidan extracted from Sargassum species accounts for a relatively high percentage, only following fucose content. Meanwhile, fucoidan samples extracted from Turbinaria decurrens had galactose content almost equal to half that of fucose. In addition to two main components of fucose and galactose, all fucoidan samples of the studied seaweeds have other simple sugars with lower levels of mannose, xylose and glucose. The content of these sugar substrates varies according to each genus and each species of seaweed, but they change little in the same genus. These results are also completely consistent with previous publications on the diversity of the chemical composition of fucoidan. In addition to the compositions of sugar substrates, fucoidan molecule also contains sulfate and uronic acids bases. The sulfate content of various fucoidan samples is not much different, ranging from 21.9 7 to 25.6% compared to the number of analyzed samples, in which the highest was fucoidan of Sargassum binderi (25.6%) and the lowest was fucoidan extracted from Sargassum duplicatum (21.9%). This shows the diversity of chemical composition of fucoidan in different seaweeds, even in the same genus of Sargassum in Vietnam and in Brazil also have a very different composition. In general, the composition of fucoidan of brown seaweeds grown in temperate seas is relatively simple, mostly containing only one fucose base and a very small amount of other simple sugars. Whereas, component of fucoidan of seaweed in tropical seas in general and in Vietnamese waters in particular is mainly in the galactofucan group, mainly composing of fucose and galactose with a small amount of other sugar bases such as rhamnose, xylose, mannose, glucose, etc. And the difference in compositions and content of single sugars of fucoidan from different algae species once again confirms that environmental conditions have a great influence on the polysaccharide biosynthesis of brown seaweed. 4.3. Study on the structural characteristics of fucoidan from two brown seaweed species S. binderi and S. duplicatum In this thesis, we make a preliminary investigation on some structural characteristics of fucoidans from two brown seaweed species of Sargassum binderi and Sargassum duplicatum. The extraction process, the isolation efficiency and the compositions of the total fucoidans of the two brown seaweed species are described in part 4.2 of the thesis. The results are presented in Table 4.1 and Table 4.2. 4.3.1. Content and research results of Sargassum binderi Fucoidan from Sargassum binderi with hightest sulfate and lowest uronic acid content was chosen to futher monosaccharides composition analyse. Fucoidans fractionated by ion-exchange chromatography on DEAE-Mcro-Prep column using aqueous sodium chloride as eluent. As a result four fractions of fucoidan from Sargassum binderi were obtained (Fig. 4.3), yield and monosaccharides composition of these fractions were shown in Table. 4.3. These results revealed that the component of fucoidan extracted from Sargassum binderi was heterogeneous with respect to not only molecular weight and sulfate contents, but also sugar constituents. These fractions were characterized as sulfated galactofucan, similar results have been reported for fucoidans from other brown seaweeds. So that structural analysis of fucoidan is very complex, we will use a representative fraction with simple component to study structural characteristic of fucoidan. 13C-NMR spectrum of fucoidan fraction F2 isolated from Sargassum binderi shown in Fig.4.4. It contented several intense signals between in the anomeric (99.7-103.18 ppm) and the high-field (15.40 ppm and 16.16 ppm) regions, which are typical of α-L-fucopyranosides. Signals in the region 67-84 ppm were attributed to C2-C5 carbons of the pyranoid ring. The signals at 61.5 ppm (CH2OH of β-Dgalactopyranose) and 65.0 ppm (CH2OR of β-D-galactopyranose) were attributed to non-6-linked and substituted at O6 β-D- galactopyranose residues in fucoidan F2. Some intense signals at 173.7-174.6 ppm and 20,92 ppm confirmed the presence of O-acetyl group. The 1H-NMR spectrum was also resolved satisfactorily (not shown). It included several intense signals in the α-anomeric (5.09-5.23 ppm) and high-field (1-1.5 ppm). In additon, the signals at 2-2.2 ppm confirmed the presence of O-acetyl group. 4.3.2. Content and research results of Sargassum duplicatum The results of determining the chemical composition (Table 4.4) show that SDAuF1 and SDAuF2 fractions have fucose and galactose which are major neutral sugar compositions. Fucose of the SDAuF2 fraction is higher (59.5%) than SDAuF1 fraction (40%), SDAuF2 fraction does not have mannose. Both fractions do not have glucose, xylose content of SDAuF2 fraction only accounts for a small amount. 8 Total FSDu fucoidan was isolated from brown seaweed S. duplicatum with the efficiency of 2.28% according to the method of Bilan et al. The IR spectrum shown in Figure 4.6 of FSDu appeared an absorption signal of 1.244 cm-1 (the unique oscillation of S=O bond). The characteristic signal range for C-O-S bond at 800-845 cm-1 is not clearly shown. Similar to a lot of fucoidan extracted from other brown seaweed in the world, FSDu fucoidan from S. duplicatum also has a very complicated 1H-NMR spectrum due to the large number of peaks stacked on each other (Figure 4.7). However, 1H- NMR spectrum also has unique resonant signals facilitating in recognizing fucoidan. These are signals that appear in the proton anomer reg