Summary of chemistry doctoral thesis: 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.

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

Supervisor 1: Asc.Prof.Dr Bui Minh Ly

Supervisor 2: Asc.Prof.Dr Nguyen Quyet Chien

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 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

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),

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

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 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

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-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

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 và 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 5 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

 For Turbinaria genus, we chose Turbinaria decurrens because there has no scientist studies its complete

structure

 For Sargassum genus, we chose Sargassum aquifolium because it has not been studied in Vietnam as

well as in the world On the other hand, this fucoidan has an additional component of uronic acid This promises the high ability in finding out a newly type of fucoidan structure

4.4 Studying the structure and biological activity of fucoidan isolated from Sargassum aquifolium 4.4.1 The extraction and purification of fucoidans

Similar to other fucoidans, FSA is a mixture of various anionic polysaccharides with different forming constituents and charge density, so that they can be separated into more homogeneous fractions by the ion-exchange chromatography method Specifically, FSA was separated into fractions with ascending sulfate content on the anion exchange chromatography column as follows:

- FSA was a heterogeneous mixture of polysaccharides with different compositional characteristics

- Sulfat content of the fractions increases gradually corresponding to the concentration of the elution solution, while the content of the uronic acid diminishes

- FSA-0,5M fraction (4.7% according to FSA) with high uronic acid and glucose content can be explained

by the presence of impurities in the samples which are alginate and laminaran

- The main fraction of FSA-1,0M (21.9% according to FSA) with especially complex single-sugar components with the presence of significant amount of fucose, galactose, xylose and mannose, and sulfate groups and uronic acid

- FSA-1,5M fraction (9.8% according to FSA) is composed mainly from fucose, galactose and sulfate Other components are few in content

- FSA-2M fraction (2.3% according to FSA) has the simplest composition, almost including fucose,

galactose and sulfate However, its content in FSA is too little

4.4.2 Determining the structure of fucoidan

4.4.2.1 Desulfation

To elucidate structures of the polysaccharides, fractions FSA-1.0M and FSA-1.5M were desulfated using the well-known solvolytic procedure, by heating of pyridinium salts of polysaccharides in a DMSO-MeOH mixture Desulfated preparations were isolated by dialysis Some degradation of the polysaccharides was observed and accounted for by preferential split of labile fucosidic linkages Desulfated preparation 1.0MdeS, that contained a minor amount of residual sulfate, was obtained in a rather high yield of 56.6% Total monosaccharide content in 1.0MdeS increased as a result of elimination of sulfate, but the relative fucose content decreased evidently owing to partial cleavage Similar compositional changes occurred in 1.5MdeS, which was obtained in a yield of 44.0% (Table 4.6)

Table 4.6 Yields and composition (in %) of FSA and fractions obtained after anion-exchange

chromatography and desulfation 1,0M-deS and 1,5M-deS (b % of FSA)

Fucoidan H% Fuc Xyl Man Glc Gal Uronic acid SO3Na

FSA-1.0M 21.9b 15.9 5.9 3.5 2.2 11.3 13.6 21.8

FSA-1.5M 9.8b 19.2 4.6 2.2 1.7 25.5 5.3 29.2

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 CH2OH 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

tri-substituted 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