The research goals of the dissertation: The research separates, identifies structural characteristics and tests biological activity of fucoidan 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.
MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY 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 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 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 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, pages of introduction, 45 pages of overview, 21 pages of research methods, 15 pages of experiment, 79 pages of result and discussions, pages of conclusions and recommendations, pages of publication list, and 12 pages of references Chapter 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 brown seaweed species including Sargassum polycystum (Fsp), 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 above-mentioned species Chapter 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 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 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 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 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) 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 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 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 13 C-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-Dgalactopyranose 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 not have glucose, xylose content of SDAuF2 fraction only accounts for a small amount 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 region (5.0-5.5 pm) and signals in high field region (1.2 - 1.5 ppm) of CH3 group of α-L- Fucopyranose ring On the 1H-NMR spectrum of fucoidan from S duplicatum, there also appears unique signals that help us in recognizing fucoidan These are signals in the regions of 1.2-1.4 ppm (methyl group of fucose) and 5.1 -5.3 ppm (H-anomer of →3)-α-L-Fucp (1→) The signals at 5.3 ppm and 3.5 ppm characterized for H-1 and H-6 of β-D- galactose residue In addition, the signal at 2.1 to 2.3 ppm also confirmed the presence of the O-acetyl group in the molecule of this fucoidan In an independent study with this thesis, the exact structure of FSDu fucoidan was determined by Usoltseva et al at Russian Academy of Sciences in the Far East Accordingly, this fucoidan is a sulfated and acetylated galactofucan FSDU has a very complicated branching structure that makes its NMR spectrum very difficult to explain Its main chain is made up primarily of the 4)-α-L-Fuc-(1 β-D-Gal alternately bonding each other Its main chain connects with galactose O-6 and is formed by fucose bases with chain connection at O-3 and sulfate substituents at O-2 and O-4 positions The branching chain has up to sugar residues or possibly more General conclusions for studying content of 4.2 and 4.3 in order to choose the subject of brown seaweed species for in-depth study of the structure and activity: The analysis results of fucoidan and other water-soluble polysaccharide components show that the fucoidan of algae species of different genus are different, the species of seaweed of the same genus or the same species are also different in its composition and mole ratio between single sugar bases This shows that the composition as well as the structural characteristics of fucoidan are extremely complex However, fucoidan of the above-mentioned algae species has a common characteristic that in addition to high sulfate content, fucose and galactose always account for higher amount compared to others for which they are known as sulfated galactofucan According to published documents, fucoidan of brown seaweed species in general and sulfated galactofucan in particular possess a very wide and diversified biological activity spectrum such as anticancer, anticoagulant, anti-viral, Fucoidans extracted and isolated from brown seaweed Sargassum feldmannii, Sargassum duplicatum, Sargassum denticarpum and Sargassum binderi are sulfated fucogalactan containing sulfate esters and uronic acid groups, along with major sugars of fucose and galactose, and a small amount of single sugars of mannose, xylose and glucose The structure of fucoidan extracted from Sargassum duplicatum and Sargassum binderi was thoroughly investigated After studying the chemical composition of fucoidan from some brown seaweed species, we chose among species of each Sargassum, Turbinaria genus each typical one for in-depth study of the structure and biological activity These include Sargassum aquifolium and Turbinaria decurrens which are also popular brown seaweed species in Vietnamese seas in general and in Nha Trang Bay in particular 10 4.4.2.2 NMR spectroscopy of FSA NMR spectra provide valuable information on polysaccharide structures, but application of this method to algal fucoidans is limited by complexity of their molecules There are only few examples of direct elucidation of an intact fucoidan structure by NMR spectroscopy A more useful approach is a combination of comparative NMR and chemical analyses of intact and desulfated polysaccharides In the particular case of FSA and its fractions, the NMR spectra were too complex to be directly interpreted Even the 13C NMR spectrum of fraction FSA-2.0M having the simplest composition (sulfated fucogalactan) contained at least six signals in the anomeric region at 105-95 ppm and the same number of signals for methyl groups of fucose residues at 20-17 ppm Signals for unsubstituted CH2OH groups of galactose were absent These data suggested that fraction FSA-2.0M possessed a highly branched irregular structure with different sulfate positions and different linkages between the monosaccharides As expected, the 13C NMR spectrum of more compositionally diverse fraction FSA-1.5M was more complex It showed additional signals in the anomeric region and resonances of unsubstituted CH2OH groups at 63-61 ppm Noteworthy, desulfation did not significantly simplify the spectra The 13C NMR spectrum of fraction FSA-1.0M also was complex With one predominated signal at 17.0 ppm in a group of methyl resonances of fucose residues, the anomeric region at 110-95 ppm contained >10 signals of similar intensities Signals for CH 2OH at 63-61 ppm and COOH of uronic acids at 175.6 ppm also were present Proton spectra of all fractions were resolved unsatisfactorily that precluded application of 2D 1H/13C NMR spectroscopy for assignment of signals 4.4.2.3 Methylation analysis As a result, three derivatives of xylose, nine derivatives of fucose, and 16 derivatives of hexoses (mannose and galactose) were found (Table 4.7) Interestingly, several methylated fucitols were derived from fucofuranose residues Earlier, a considerable amount of fucofuranose has been found in only one fucoidan isolated from Chordaria flagelliformis Sulfated preparations contained mainly di- and even trisubstituted fucose residues, which, being components of linear chains, should therefore additionally carry sulfate groups or branches Desulfated samples contained many terminal nonreducing fucose residues probably linked to cores built up of other monosaccharides The mannose content in 1.5MdeS was negligible, and hence, all the methylated hexitols were derived from galactose residues, which were predominantly 6- and 4-linked The parent fraction FSA-1.5M contained some 6-linked galactose residues devoid of other substituents, but most of them were additionally substituted, probably with fucose or sulfate Fraction 1.0MdeS contained similar amounts of mannose and galactose Methylated derivatives of these monosaccharides could not be distinguished from each other by mass spectra that further complicated interpretation of methylation results of samples FSA-1.0M and 1.0MdeS Table 4.7 Methylation analysis of sample FSA Position of O-Me Inferred positions FSA-1.0M , 1.0MdeS, FSA- groups in mol% 1.5M of substitution mol% 1.5MdeS, , mol% mol% Xyl: 2,3,4 Xylp→ 10 2,4 →3Xylp→ - tr 11 2,3(3,4) tr 3* Fuc: 2,3,5 Fucf→ 1 2,3,4 Fucp→ 14 2,3 →4(5)Fucp(f)→ 6 3,5 →2Fucf→ - - - 2,4 →3Fucp→ 6 3** →3,4(5)Fucp(f)→ 11 →2,4(5)Fucp(f)→ 7*** - →2,3Fucp→ - + - Fuc →2,3,4Fuc→ - - 2,3,4,6-Man Manp→ tr tr 2,3,4,6-Gal Galp→ 12 3,4,6 →2Hexp→ - - - 2,3,6 →4Hexp→ 10 15 2,3,6 →4Hexp→ 2,4,6 →3Hexp→ - 2,3,4 →6Hexp→ 21 2,6 →3,4Hexp→ - 4,6 →2,3Hexp→ 3 - tr Hex: 3,6+4,6 3,6 →2,4Hexp→ 3 tr 2,3 →4,6Hexp→ 2 2,4 →3,6Hexp→ 10 - - 3,4+2,4 →3,4,6Hexp→ tr →2,3,6Hexp→ - →2,4,6Hexp→ - 3(4) 10 - 12 3(4) Gal - 2 4.4.2.4 Fractionation of 1.0M-deS Desulfated preparation 1.0M-deS contained uronic acids and hence retained the anionic properties It was fractionated into six fractions deS-1 to deS-6 using anion-exchange chromatography on DEAESephacel Their yields and composition are given in Table 4.8 The fractions were characterized by methylation analysis (Table 4.7) and NMR spectra Table 4.8 Yields and composition (in %) of FSA and fractions obtained after anion-exchange chromatography and desulfation (c % of 1.0MdeS) Fucoidan Yields Fuc Xyl Man Glc Gal % FSA-1.0M 21.9 c 1.0MdeS deS-1 3.1 c Uronic SO3Na acid 15.9 5.9 3.5 2.2 11.3 13.6 21.8 8.1 4.0 7.8 4.3 14.0 30.6 2.8 5.7 15.9 5.4 8.0 30.1 2.5 n.d deS-2 13.8 c 15.4 12.1 2.3 3.3 38.8 7.1 2.3 deS-3 15.4 c 16.4 10.1 4.5 5.1 24.1 12.1 1.9 deS-4 34.6 c 6.7 2.4 8.4 2.9 4.2 40.9 n.d 13.8 c 11.4 3.7 4.4 3.1 5.3 47.0 1.5 7.4 2.6 4.0 2.7 3.0 51.1 1.5 deS-5 deS-6 7.6 c 4.4.2.5 Structural characteristics of deS-2 and deS-3 fractions The most intense signals in the anomeric region of the 1H-NMR spectrum of deS-2 were at δH = 5.31ppm (doublet, Hz), 4.49, 4.47 and 4.45 ppm (doublets, Hz) COSY, TOCSY and ROESY experiments showed that these signals belonged to α-Galp, β-Xylp, and β-Galp, respectively 2D 1H/13C HSQC spectrum (Fig 4.15 and Table 4.9) enabled assignment of the major signals in the 13C NMR spectrum of deS-2 Fig 4.15 HSQC spectrum of deS-2 13 Fig 4.16 Structural fragments in desulfated preparation deS-2 Table 4.9 1H- and 13C-NMR chemical shifts of deS-2 H-1 H-2 H-3 H-4 H-5 H-6 C-1 C-2 C-3 C-4 C-5 C-6 5.31 3.88 3.96 4.25 4.18 3.85,385 101.7 70.6 70.0 80.0 73.0 61.9 4.45 3.81 3.77 4.15 3.73 3.77,3.77 104.4 71.7 81.6 69.6 76.3 62.2 4.45 3.81 3.77 4.11 3.80 4.06,3.97 104.4 71.7 81.6 69.7 74.2 70.7 4.49 3.30 3.48 3.65 4.00,3.32 - 102.9 74.2 77.0 70.5 66.4 - 4.47 3.32 3.58 3.80 4.12,3.40 - 104.6 74.0 75.0 77.5 64.2 - 4.49 3.56 3.69 4.02 3.98 4.04,4.04 deS-2 A B C 4)-α-D-Gal -(1 3)- -D-Gal -(1 3,6)- -D-Gal -(1 D - -D-Xyl -(1 E F 4)- -D -Xyl -(1 6)- -D-Gal -(1 104.6 72.1 74.0 70.5 74.7 71.8 All α-Galp residues were 4-linked, β-Galp residues 3- or/and 6-linked, and β-Xylp residues either 4linked or occupied non-reducing termini of side chains ppm 1D/6C 1А/3А 70 72 1А/5А 74 76 1E/4E 1B/4А 1D/4E 78 80 1А/3B 82 Fig 4.17 HMBC spectrum of deS-2 14 Structure of the polymer was inferred by analysis of the 2D 1H/13C HMBC spectrum (Fig 4.17), which showed the following inter-residual correlations: H-1(α-Galp, A)/C-3(β-Galp, B and C), H-1(β-Galp, B)/C-4(α-Galp, A), H-1(β-Xylp, D)/C-6(β-Galp, C), H-1(β-Xylp, D)/C-4(β-Xylp, E), and H-1(β-Galp, F)/C6(β-Galp, F) (a minor component) These data suggested the presence of a core built up of / 4)-α-Galp(1→3)-β-Galp-(1→ units, in which a part of β-Galp residues were substituted with single β-Xylp or short (1→ 4)-β-Xylp chains ranging from a disaccharide The fucose and xylose contents of deS-2 were similar, but an attempt to determine positions of the fucose residues by NMR spectroscopy failed The 2D NMR spectra of deS-2 indicated the presence of a minor (1→6)-β-Galp homopolymer It may either be attached as a side chain, side by side with xylose, at position of 3-linked β-Galp of the core or represent a separate minor polysaccharide, which is not connected to the main polymer Methylation analysis (Table 4.7) confirmed the presence of a core of alternating 4- and 3-linked Galp residues bearing branches at position In addition, there were identified i) minor 6-linked Galp, ii) variously linked Fuc (both fucopyranose and fucofuranose), and iii) terminal and 4-linked Xylp Methylation analysis showed that polysaccharides of deS-3 had similar structures but they i) contained more 4-linked Gal and less 3-linked Gal, ii) 6-linked Gal residue were almost absent, and iii) terminal Xyl predominated over 4-linked residues Exact structures of these branched polysaccharides remain to be determined Earlier, various fucogalactans have been reported in several other brown algae, such as Undaria pinnatifida and Laminaria japonica 4.4.2.6 Structural characteristics of deS-4 fraction Similar analysis of 2D NMR spectra of deS-4 enabled identification of the main polysaccharide with a core of alternating 2-linked α-D-Manp and 4-linked β-D-GlcpA About a half of the mannose residues carried at position side chains of single α-L-Fucp or single β-D-Xylp in a ratio of ~4:1 Sequence of the monosaccharide residues in the core was confirmed by the HMBC spectrum (Fig 4.19), which showed the following correlations: H-1(α-Manp, S)/C-4(β-GlcpA, R), H-1(α-Manp, U)/C-4(β-GlcpA, T), H-1(β-GlcpA, R)/C-2(α-Manp, U) and H-1(β-GlcpA, T)/C-2(α-Manp, S) Location of α-Fucp and β-Xylp at O-3 of α-Manp followed from H-1(α-Fucp, V)/C-3(α-Manp, U) and H-1(β-Xylp, G)/C-3(α-Manp, U) correlations, respectively These data were confirmed by methylation analysis Earlier, similar fucoglucuronomannans have been found in several other brown algae Particularly, a polysaccharide from Kjellmaniella crassifolia consists of branched trisaccharide repeating units (similar polysaccharides are probably present in Saccharina latissima and Laminaria bongardiana Recently, it has been shown that short fucooligosaccharide side chains, side by side with single Fucp, are present in a fucoglucuronomannan from Kjellmaniella crassifolia Yet, single side-chain Xylp residues are reported in such polysaccharides for the first time 15 Fig 4.18 HSQC spectrum of deS-4 Fig 4.19 HMBC spectrum of deS-4 Fig 4.20 Structural fragments in desulfated preparation deS-4 16 Table 4.10 1H- and 13C-NMR chemical shifts of deS-4 deS-4 H-1 H-2 H-3 H-4 H-5 H-6 C-1 C-2 C-3 C-4 C-5 C-6 5.38 4.14 3.81 3.69 3.69 3.84,3.79 100.0 79.0 70.9 68.0 74.0 61.5 5.36 4.32 3.89 3.83 3.74 3.84,3.79 99.8 74.6 74.7 66.0 74.0 61.5 5.36 4.34 3.84 3.80 3.74 3.84,3.79 99.8 73.9 76.5 65.9 74.0 61.5 4.46 3.38 3.65 3.78 3.75 - 102.8 74.2 77.5 78.8 77.6 176.0 4.46 3.38 3.65 3.78 3.75 - 103.0 74.2 77.5 78.0 77.6 176.0 5.08 3.79 3.93 3.81 4.22 1.20 96.4 69.2 70.5 73.1 67.9 16.6 4.38 3.30 3.44 3.62 4.00,3.29 - 104.6 74.4 4.4.2.7 Structural characteristics of deS-6 fraction 77.0 70.6 66.5 - S Uv UG T R 2)- -D-Man -(1 - -D-Man -(1 2,3)- -D-Man -(1 - -D-Gle A-(1 4)- -D-Gle A-(1 V -L-Fuc -(1 G -D-Xyl -(1 HSQC spectrum and HMBC spectrum have been showed on Fig.4.21 and Fig.4.22 Fig 4.21 HSQC spectrum of deS-6 17 Fig 4.22 HMBC spectrum of deS-6 Fraction deS-6 was distinguished by a higher uronic acid content and, according to NMR spectra (Fig 4.21), contained a glucuronan as the main component It had a core of 3-linked β-D-GlcpA residues, a part of which carried single β-D-Xylp or single α-L-Fucp (minor) at position Structure of the core was confirmed by an intense β-GlcpA H-1/C-3 correlation peak in the HMBC spectrum (Fig 4.22) Position of αFucp and β-Xylp at O-4 of β-GlcpA followed from intense H-1(β-Xylp, K)/C-4(β-GlcpA, YK) and H-1(αFucp, H)/C-4(β-GlcpA, YH) (minor) correlation peaks A similar fucoglucuronan has been reported as a minor component of fucoidan from Saccharina latissima In addition, deS-6 contained a marked amount of fucoglucuronomannan, which was the main component of deS-4 Fig 4.23 Structural fragments in desulfated preparation deS-6 Table 4.11 1H- and 13C-NMR chemical shifts of deS-6 18 4.4.2.8 Structural characteristics of deS-5 fraction Fraction deS-5 was essentially a ~1:1 mixture of fuco(xylo)glucuronomannan and xylo(fuco)glucuronan, the main components of deS-4 and deS-6, respectively 4.4.2.9 Study some structural characteristics of FSA-1.5M fraction by methylation analysis method in combination with ESI-MS/MS spectra All data obtained enables us to derive the overall structural characteristics for fucoidan of FSA-1.5M fraction as follows: - FSA-1.5M fucoidan has a high content of sulfate (31.5%), has mainly composed of α-L-fucose (19.2%) and galatose (5.5%) Fucose was found in both pyranose and furanose rings Galactose is only in pyranose ring - FSA-1.5M fucoidan has a highly branching structure The main chain was formed mainly by fucose residues that alternatively links at O-3 and O-4 positions Galactose residues was located at the non-reducing part and links with O-3 of fucose forming branching chains - In the core structure, sulfate groups are mainly located at O-2 position on both fructose and galactose residues 4.4.3 Biological activities 4.4.3.1 Cytotoxic activity Human cancer cell lines HepG2 (hepatocellular carcinoma), LU- (lung adenocarcinoma), and RD (rhabdomiosarcoma) were used for examination of the cytotoxicity of FSA fractions The cells were cultured as monolayers, and cytotoxic assay was performed according to the method employed at the National Cancer Institute, USA (NCI) Ellipticin was used as positive control Cytotoxicity was presented as percentage of cells surviving at a given polysaccharide concentration (Table 4.13) The most active fucoidan fractions FSA-1.5M and FSA-2.0 M guaranteed 50% inhibition of Hep-G2 cancer cell growth at concentrations IC50 of 60.7 and 46.8 µg/mL, respectively Table 4.13 Survival of cancer cells in the presence of FSA fractions Number Sample Surviving cells (%) Concentration (g/mL) Hep-G2 LU-1 RD FSA-1.5M 100 40,50,8 41,20,3 70,21,3 FSA-2.0M 100 29,30,5 78,250,2 40,51,3 Ellipticin 0,50,5 1,10,2 0,030,0 4.4.3.2 In vitro anti-tumor activity In vitro antitumor assay was performed as described Inhibitory action of fucoidan fractions on colonies formation by Hep-G2 cells was assessed by soft agar assay and presented as density (%) or size (mm) of colonies related to control (Table 4.14, Fig 4.28) Microphotographs demonstrated changes in dimension and morphology of colonies Therefore, fucoidan fractions induced effective inhibition of the tumors, probably by apoptosis of Hep-G2 cancer cells Fractions 1.5 M and 2.0 M decreased the density of colonies on soft agar by 24.72% and 34.03% and average colony dimensions by 22.14% and 41.11%, respectively, compared to ellipticin control 19 Table 4.14 Influence of fractions 1.5M and 2.0M on the average dimension and density of Hep-G2 Dimension of colonies Decrease Concentration Sample Diameter (µm) (g/ml) diameter (%)(Compared of Decrease of density of in colonies in % to (Compared to control) control) Control (-) 41,35 1,85 0 1.5M 100 31,13 1,42 24,72 22,14 1,12 2.0M 100 27,28 0,95 34,03 41,11 0,88 1,5M (100 g/ml) Control (-) 2,0M (100 g/ml) Fig 4.28 Microphotographs demonstrating influence of fucoidan fractions on Hep-G2 cell growth 4.4.3.3 Anticoagulant activity Heparin-like anticoagulant properties of FSA fractions related to the standard low-molecular heparin preparation (enoxaparin) were examined by the activated partial thromboplastine time (APTT) test Fraction 0.5 M was inactive, whereas the anticoagulant properties of other fractions increased from 1.0 M to 2.0 M with the sulfate content 2APTT values (concentrations of polysaccharides leading to two-fold delay of clot formation, 6.5 ± 0.4 µg/mL for 2.0 M and 3.9 ± 0.4 µg/mL for enoxaparin) showed that even the most Time of clot formation (s) sulfated fraction 2.0 M was about half as active as enoxaparin (Fig 4.29) Concentration (µg/ml) Figure 4.29 Anticoagulant activity of FSA fractions (test APTT) 20 Summary of study results of content 4.4: From brown seaweed Sargassum aquifolium in Nha Trang seas, it has been extracted a total fucoidan called FSA with an efficiency of 3.7% calculated on dry material The FSA has high average molecular weight (110 kDa) and complex structure with major components including L-Fucose, D-galactose, D-mannose, D-glucuronic acid, D-xylose and sulfate The study of FSA structure directly by MS and NMR spectrum methods is infeasible FSA was separated into four fractions (denoted FSA-0.5M, FSA-1.0M, FSA-1.5M, and FSA-2.0M, respectively) with ascending sulfate content by anion exchange chromatography method The main fractions of FSA-1,0M and FSA-1,5M (21.0 and 9.8% from FSA) are analyzed by methylation method prior to and after desulfation The desulfation products of FSA-1.0M, which is 1.0M-deS was separated into six fractions (denoted from deS-1 to deS-6) with ascending uronic acid content by anion exchange chromatography method Their structural characteristics are determined by methylation analysis method and by NMR data It has resulted in the identification of three polysaccharides with different basic structures: + First polysaccharide includes a main chain of 2)-α-D-Gal-(1 and 4)-β-D-GlucA-(1 linked alternately About half of 2)-α-D-Gal-(1 with substitutes of α-L-Fucp or β-D-Xylp in O-3 + Second polysaccharide has one main chain which is a (13)-β-D-glucopyranuronan partially substituted by a single β-D-Xylp or α-L-Fucp residue at O-4 + Third polysaccharide has is the main chain of a xylo(fuco)galactan with a line constituted from 4)α-D-Gal-(1 and 3)-α-D-Gal-(1 residues linked alternately 3)-α-D-Gal-(1 residues carries a substituent of a β-D-Xylp or a short of 4)-β-D-Xylp-(1, 6)-α-D-Gal-(1 and α-L-Fuc residues with linked in different positions + In FSA, these polysaccharides carry sulfate substituents at different positions, with no definite rules FSA-1,5M chromatographic fraction with high content of sulfate (31.5%) with simple component, mainly from a-L-fucose (19.2%) and galatose (5.5%), but with complex structure, was not directly studied by MS and NMR spectra Study of FSA-1,5M by methylation analysis combined with ESIMS/MS spectrum of partial hydrolysis product with TFA shows that: FSA-1,5M fucoidan has mainly constituted of a-L-fucose (19.2%) and D-galatose (5.5%) Fucose was found in both pyranose and furanose rings Galactose was only in pyranose ring FSA-1,5M Fucoidan has a highly branching structure The main chain was formed mainly by fucose residues that alternatively links at O-3 and O-4 positions Galactose residues were located at the non-reducing part and links with O-3 of fucose forming branching chains In the core structure, sulfate groups are mainly located at O-2 position on both fructose and galactose residues The results showed that this FSA-1,5M fraction is a galactofucan, its main chain was composed of alternating -(1 → 3)-linked and -(1 → 4)-linked fucose residues, the galactose residues were in branch and connected to fucose by linkage (1→6) This fraction was investigated for cytotoxicity activity and the results showed that it inhibits both two human cancer cell lines Hep-G2 and RD FSA fucoidan fractions have anticoagulant, cytotoxic and anti-tumor activity These activities increase corresponding with the fucose content and sulfated level 21 4.5 Structural determination and cytotoxic activity of fucoidan extracted from brown seaweed Turbinaria decurrens - Total fucoidan (FTD) was isolated with an efficiency of 2.19% FTD has an average molecular weight of 227 kDa, with sulfate content of 24.3% and simple sugar components consisting mainly of fucose and galactose at a ratio of 1: 0.5 - Using DEAE-Sephadex column chromatography, fucoidan was fractionated into three fractions FTD0.5N, FTD-1.0N and FTD-2.0N with the respective efficiencies of 20, 30 and 46% - FTD-2,0N has an average molecular weight of 227 kDa, with sulfate content of 34.5% and simple sugar compositions, including fucose and galactose at a ratio of 1: 0.65 - The results of IR, NMR (1D and 2D) and ESI-MS/MS spectra showed that FTD-2.0N was a galactose fucan which composed of (1→3)-α-L-fucose-2-sulfate and -D-galactose-6-sulfate and galactose residues connect to fucose by linkage 1→4 The basic structure of FTD-2.0N fraction was proposed as follows: [→3-α-L-Fucp2(OSO3-)-(1→4)-β-D-Gal6(OSO3-)-1→]n - FTD-2.0N can inhibit tumor formation of HepG2 at concentration of 100 pg/ml 4.6 Studying size and shape of fucoidans isolated from brown seaweed in Vietnam and their relationship with cytotoxic activity - Having extracted, isolated, identified constituent components and average molecular weight of the fucoidans from 06 brown seaweed species of Nha Trang, namely: S.polycystum (Fsp), S.mcclurei (Fsm), S oligocystum (Fso), S.denticarpum (Fsd), S.swatzii (Fss), and T.ornata (Fto) - The shape and size of fucoidan samples were studied by small angle X-ray scattering (SAXS) measurements The results of the Kratky plot analysis of small-angle X-ray scattering revealed that rods like fucoidans with bulky branching chains and varying branching degree The calculated results for theoretical models show that branching chains are capable of being adjacent together and up to monosaccharides residues in length - Analysis results of Guinier plot, we can estimate the cross-sectional radius of gyration of rod-like particle (Rgc,) of the molecule of each fucoidan sample The Rgc of our fucoidans estimated ranged from 0.78 to 1.51nm suggesting a branched conformation with different branch's length of those fucoidans - The cytotoxic activity of fucoidans on Hep-G2 and RD cancer cell were tested The results showed that the samples all showed activity Two samples with highest activity was Fto (IC50 for Hep-G2 and RD was 3.1 and 1.6 µg/mL) and Fsp (IC50 for Hep-G2 and RD was 5.5 and 5.7 µg/mL) - Preliminarily surveying the relationship between cytotoxic activity and some constituent and structural characteristics of fucoidan samples showed that: a) the average molecular weight was most significantly related to the cytotoxic activity The samples with the highest average molecular weight also had the highest activity The samples with the lowest average molecular weight also had the lowest activity; b) The sulfated level of the sugars also exhibits a certain degree corresponding with cytotoxic activity High sulfate/sugar ratio is associated with high activity and vice versa; c) There was no clear correlation between the bulky level (length) of the branching chains (shown by the cross-sectional radius of gyration of rod-like particle Rgc) with cytotoxic activity The above data suggest that high molecular 22 weight and branching structures with high sulfate/sugar ratio are essential factors for cytotoxic activity of fucoidan samples Cytotoxicity for cancer cells of fucoidans isolated from brown seaweed species were investigated All fucoidans showed cytotoxic activity against the RD (Rhabdomyosarcoma) and HepG2 (hepatocellular carcinoma) cells From small angle X-ray scattering (SAXS) measurements, fucoidans have branched structure as indicated by Rgc values ranged from 0.78 to 1.51nm Our results supported the branched architecture with many sulfate groups reveals bioactivity of fucoidan but the relationship between the length of branch and the level of bioactivity was not clear CONCLUSION Studying the structure and biological activity of fucoidan from some brown seaweed species in Vietnam, we have obtained following results: The content and chemical composition of fucoidan from 11 species of brown seaweed belonging to two genera of Sargassum and Turbinaria grown in Nha Trang bay were investigated This result shows a more comprehensive view of chemical composition and structural characteristics of fucoidan Based on these results, fucoidans isolated from Sargassum aquifolium and Turbinaria decurrens have been selected for indepth study of chemical structure and biological activity The structural characteristics of fucoidan from two seaweed species of Sargassum duplicatum and Sargassum binderi were studied The results showed that fucoidans from these two species was galactofucan sulfate This result once again contributes in determining that most of fucoidans isolated from brown seaweed grown in tropical regions are commonly galactofucan sulfate From Sargassum aquifolium isolated, determined the structure and biological activity of fucoidan including FSA-0, 5M, FSA-1,0M, deS-1, deS-2, deS-3, deS-4, deS-5, deS-6, FSA-1,5M and FSA-2,0M - The structure of fucoidan was studied firstly in the world and Vietnam as xylo(fuco)galactan (deS-2), fuco(xylo)glucuronomannan (deS-4) and xylo(fuco)glucuronan (deS-6) Specially, the structure of the main chain of fucoidan fraction containing manose (deS-4), uronic acid (de-S6) and xylose (de-S2) This is a new content in the structure of fucoidan extracted from brown seaweed species in Nha Trang sea Studies on other seaweed species showed that most fucoidans are galactofucan - Biological activity results: FSA-0,5M, FSA-1,0M, FSA-1,5M, FSA-2,0M fractions possess anticoagulant, cytotoxic and prevent the development of tumor, particularly anticoagulant activity, which was first investigated on fucoidan extracted from brown seaweed in Vietnam FTD2.0N fucoidan was isolated from Turbinaria decurrens is a galactofucan sulfate formed from two sugars (1→3) –α-L-Fuc with sulfate substituent at C-2 position and sulfated α-D-galactose in C-6 position with mole ratio of L-Fuc: D-Gal = 1: 0.65 Galactose linked to fucose via glucoside 1→4 linkage FTD2.0N fucoidan from brown seaweed was also able to inhibit the formation of tumor of Hep-G2 Galactofucan sulfate (FTD2.0N) was the first fucoidan isolated from brown seaweed Turbinaria decurrens By SAXS method, we have identified the relationship between branching structure and cytotoxic activity toward cancer cells of fucoidan isolated from brown seaweed species in Vietnam The results indicate that branching structure of fucoidan with many sulfate groups, showing that fucoidan has higher biological activity than other fucoidans The effect of structural parameters (sulfate content, average molecular weight 23 and branching chain) on the biological activity of fucoidan was investigated This is a new point compared to domestic studies on the same field From the above results, we can point out following remarks: Unlike fucoidan originated in temperate waters, most of fucoidans isolated from brown seaweed in Vietnam has very complex structure and formation However, by the clever combination of classical research methods with modern tooling methods, especially two-dimensional nuclear magnetic resonance methods, multiple-mass spectral methods of MS/MS and small-angle X-ray scattering method, current science helps further clarify their delicate structure The study results of this thesis as well as of many scientific papers recently published in Vietnam show that brown seaweed species of Vietnam are not only a highly valuable marine resource with high using value but also a treasure of natural compounds with new, complex and diverse structures that are worth indepth studying NEW CONTRIBUTIONS OF THE PHD THESIS For the first time, the extraction, fractionation, isolation and structure determination of fucoidans from the brown seaweeds Sargassum aquifolium and Turbinaria decurrens distributed in Nha Trang, Vietnam were performed using modern methods of carbohydrate chemistry The application of ion-exchange chromatography, desulfation, and methylation analysis lead to simpler and more homogeneous fucoidan fractions which were suitable for the determination of structural details by NMR spectroscopy and high resolution mass spectrometry From the crude fucoidan FSA extracted from S aquifolium, fucoidan fractions were isolated Their structure determination revealed basic structural types of xylo(fuco)galactan (deS-2), fuco(xylo)glucuronomannan (deS-4), and xylo(fuco)glucuronan (deS-6) which are different from the galactofucan type commonly found in fucoidans from other Nha Trang brown seaweeds From the crude fucoidan FTD extracted from T decurrens, one fraction FTD-2,0N was obtained Its structure were determined to be as follows: [→3-α-L-Fucp2(OSO3-)-(1→4)-β-D-Gal6(OSO3-)-1→]n For the first time, the conformational structure of fucoidans extracted from Nha Trang seaweed species were studied by small-angle X-ray scattering (SAXS) measurements The analysis of the Guinier plots gave Rgc values of 0.78 - 1.51 nm for the cross-sectional radius of gyration The analysis of the Kratky plots indicated rod-like, short branched chain structure Bioassays showed that the fucoidan fractions with the highest sulfate content FSA-2,0M and FTD- 2,0N extracted from S aquifolium and T decurrens, respectively, showed cytotoxic activity and inhibitory activity against tumor formation of hepatocellular carcinoma cells FSA-2.0M was shown to possess anticoagulant activity Preliminary assessment of the structure-activity relationship of fucoidans extracted from seaweed species found in Nha Trang suggested that high molecular weight, branched chain structure, and high sulfate / sugar ratio were necessary for the cytotoxic activity of fucoidans 24 RECOMMENDATIONS On the basis of the results obtained from the thesis, we have some recommendations as follows: Continuing to study the chemical structure and biological activity of fucoidan from other brown seaweed species of Vietnam in order to find new compounds with high biological activity serving as the basis for the exploitation and effective utilization of marine resources in Vietnam Continuing to study the structural-biological relationship to further clarify the impact of polysaccharide structure and its derivatives on biological activity as a basis for creating derivatives with higher activity than the natural samples PUBLISHED WORKS OF THE DISSERTATION Maria I Bilan, Nadezhda E Ustyuzhanina, Alexander S Shashkov, Thanh Thi Thu Thuy, Bui Minh Ly, Tran Thi Thanh Van, Bui Van Nguyen, Anatolii I Usov , Sulfated polysaccharides of the Vietnamese brown alga Sargassum aquifolium (Fucales, Sargassaceae), Carbohydrate Research, Volume 449 (2017) PP 23-31, https://doi.org/10.1016/j.carres.2017.06.016 (SCI) Yoshiaki Yuguchi, Bui Minh Ly, Bui Van Nguyen, Tran Thi Thanh Van and Thanh Thi Thu Thuy, Study on branched structure-physiological activity relationship of fucoidan, Chemistry letter, Vol.45 No.7 2016 p.840-842 doi:10.1246/cl.160189 (SCI) Bui Van Nguyen, Tran Thi Thanh Van, Bui Minh Ly, Nguyen Quyet Chien, Thanh Thi Thu Thuy, Maria I Milan, Anatolii I Usov, Structural characteristics and anticoagulant activity of sulfate polysaccharide from the brown alga sargassum aquifolium , Journal of Science and Technology 54 ( 2C) (2016) 360-365 Bùi Văn Nguyên, Trần Thị Thanh Vân, Bùi Minh Lý, Nguyễn Quyết Chiến, Thành Thị Thu Thủy, Study on structure and cytotoxic activity of fucoidan extracted from brown seaweed Sargassum aquifolium, Vietnam journal of chemistry 54(6e2)(2016)215-218 Bùi Văn Nguyên, Bùi Minh Lý, Võ Mai Như Hiếu, Đặng Vũ Lương, Trần Thị Thanh Vân Thành Thị Thu Thủy, Xác định cấu trúc fucoidan tách chiết từ rong nâu Turbinaria decurents phương pháp phổ IR, NMR ESIMS, Tạp chí Khoa học cơng nghệ T.52 (5B), 2014 (637-642) Bui Van Nguyen, Cao Thi Thuy Hang, Tran Thi Thanh Van, Nguyen Thi Hai Au, Dang Xuan Cuong, Natalia M Shevchenko, Svetlana P Ermakova, T.N Zvyagintseva and Pham Duc Thinh, The content and chemical composition of water-soluble polysaccharides isolated from some sargassum seaweed species in viet nam, Journal of Science and Technology 54 (2B ) (2016), pp 84-90 REFERENCES The dissertation have been used 122 references including: science paper, book, the dissertation, etc ... 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... 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... 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