Summary of chemistry doctoral thesis: Structural characteristics and biological activities of fucoidans extracted from selected vietnamese brown seaweeds

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


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.
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 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 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.

9



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 DMSOMeOH 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

8.1

4.0

7.8

4.3

14.0

30.6

2.8

19.2

4.6

2.2

1.7

25.5

5.3

29.2

12.8

5.3

2.7

1.8

31.6

10.4

4.1

1.0MdeS
FSA-1.5M
1.5MdeS

9.8

b


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→

3

10

2

2,4

→3Xylp→

2

-

tr.

4


11

2,3(3,4)

tr.

3*

4

7

Fuc:

2,3,5

Fucf→

1

1

2

1

2,3,4

Fucp→

3

14

4

7

2,3

→4(5)Fucp(f)→

6

6

3

4

3,5

→2Fucf→

-

-

3

-

2,4

→3Fucp→

6

6

3**

6

2

→3,4(5)Fucp(f)→

6

1

11

2

3

→2,4(5)Fucp(f)→

9

1

7***

-

4

→2,3Fucp→

-

3

+

-

Fuc

→2,3,4Fuc→

4

-

9

-

2,3,4,6-Man

Manp→

tr.

2

tr.

1

2,3,4,6-Gal

Galp→

1

5

2

12

3,4,6

→2Hexp→

-

5

-

-

2,3,6

→4Hexp→

4

10

2

15

2,3,6

→4Hexp→

3

4

1

2

2,4,6

→3Hexp→

4

6

1

-

2,3,4

→6Hexp→

2

4

8

21

2,6

→3,4Hexp→

7

-

7

2

4,6

→2,3Hexp→

8

9

1

3

3

-

tr.

Hex:

3,6+4,6
3,6

→2,4Hexp→

3

3

tr.

2

2,3

→4,6Hexp→

2

2

1

2

2,4

→3,6Hexp→

5

3

10

-

-

3,4+2,4
2

→3,4,6Hexp→

5

tr.

4

→2,3,6Hexp→

-

1

3

→2,4,6Hexp→

-

1

3(4)

9

3
10

2

-

12

3(4)

9

Gal

1

-

2

2

5

4

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, 3 Hz), 4.49, 4.47 and 4.45 ppm (doublets, 8 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 6 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 6. 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 3 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 4. 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- 1 (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

1

FSA-1.5M

100

40,50,8

41,20,3

70,21,3

2

FSA-2.0M

100

29,30,5

78,250,2

40,51,3

3

Ellipticin

5

0,50,5

1,10,2

0,030,0

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

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 (13)-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 6 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 5
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 6 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:
1. 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.
2. 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.
3. 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.
4. 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.
5. By SAXS method, we have identified the relationship between branching structure and cytotoxic activity

toward cancer cells of fucoidan isolated from 6 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
1.

For the first time, the extraction, fractionation, isolation and structure determination of fucoidans

from the 2 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.
2.

From the crude fucoidan FSA extracted from S. aquifolium, 6 fucoidan fractions were isolated. Their

structure

determination

revealed

3

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.
3.

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

4.

For the first time, the conformational structure of fucoidans extracted from 5 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.
5.

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.
6.

Preliminary assessment of the structure-activity relationship of fucoidans extracted from 5 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.