MINISTRY OF EDUCATION AND TRAINING
NHA TRANG UNIVERSITY

HASSAN IYUNADE HASSANAT

OPTIMISATION OF ULTRASOUND-ASSISTED
EXTRACTION CONDITIONS FOR ANTIOXIDANT AND
TYROSINASE INHIBITORY ACTIVITIES OF SOME
VIETNAMESE BROWN SEAWEED SPECIES

MASTER THESIS

KHANH HOA – 2020


MINISTRY OF EDUCATION AND TRAINING
NHA TRANG UNIVERSITY

HASSAN IYUNADE HASSANAT

OPTIMISATION OF ULTRASOUND-ASSISTED
EXTRACTION CONDITIONS FOR ANTIOXIDANT AND
TYROSINASE INHIBITORY ACTIVITIES OF SOME
VIETNAMESE BROWN SEAWEED SPECIES
MASTER THESIS
Major:

Food Technology

Topic allocation Decision:
Decision on establishing the

Committee:
Defense date:
Supervisors:
DR NGUYEN THE HAN
DR HONG NGOC THUY PHAM
Chairman of Committee:

Faculty of Graduate studies:

KHANH HOA – 2020


UNDERTAKING
I undertake that the thesis titled: Optimisation of ultrasound-assisted extraction
conditions for antioxidant and tyrosinase inhibitory activities of some Vietnamese brown
seaweed species is my work. The work has not been presented elsewhere for

assessment until the time this thesis is submitted.

Hassan Iyunade Hassanat
31/08/2020

iii


ACKNOWLEDGEMENT
All glory to my maker, the one that has made all this possible. Special thanks to
VLIR-NETWORK, Vietnam, for providing me with a fully-funded scholarship to
study in Vietnam. I will love to extend my sincere gratitude to my supervisors Dr
Nguyen The Han and Dr Thuy Pham, for their timeless advice and for guiding me

throughout the course of working on this thesis. Your immerse support will always be
remembered. Special thanks to my colleagues who made this two-year master program
fun despite its being tasking. I appreciate the members and staff of Nha Trang
University and my good friends, Funmilola, Sabine, Mule for their constant support
and encouragement. I wish to thank the Research Fund of Khanh Hoa province for
financial support (Project number: ĐT-2017-20902-ĐL).
Finally, many thanks to my parents, Mr and Mrs Hassan. I could never ask for
better parents. To my siblings, Fade and Fola, thanks for the unconditional support.
To an exceptional man, Ayo. Thanks for being the sweetest

iv


TABLE OF CONTENT

UNDERTAKING ........................................................................................................ iii
ACKNOWLEDGEMENT ............................................................................................iv
TABLE OF CONTENT .................................................................................................v
LIST OF ABBREVIATIONS .................................................................................... viii
LIST OF TABLES........................................................................................................ix
LIST OF FIGURES .......................................................................................................x
APPENDICES ............................................................................................................ xii
ABSTRACT .............................................................................................................. xiii
CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW ...............................1
1.1 Introduction ..........................................................................................................1
1.1.1 Problem statement ..........................................................................................2
1.1.2 Research questions .........................................................................................3
1.1.3 Hypothesis .....................................................................................................3
1.1.4 Prediction .......................................................................................................3
1.1.5 Main objectives ..............................................................................................3

1.1.6 Specific objectives .........................................................................................4
1.1.7 Conceptual framework ...................................................................................4
1.2. Literature review .................................................................................................4
1.2.1 Seaweed .........................................................................................................4
1.2.2 Seaweed in Vietnam.......................................................................................6
1.2.3 Brown seaweed ..............................................................................................7
1.2.4 Major antioxidant constituents of brown seaweed .........................................8
1.3 Tyrosinase .......................................................................................................... 15
1.3.1 Role of tyrosinase in melanogenesis ............................................................ 16
1.3.2 Physiological roles of melanin ..................................................................... 18
v


1.3.3 Common tyrosinase inhibitors ..................................................................... 19
1.3.4 Potential Applications of Tyrosinase Inhibitors from Brown Seaweed ........ 21
1.4 Extraction of bioactive compounds from seaweed ............................................. 22
1.4.1 Ultrasound-assisted extraction (UAE) .......................................................... 23
1.4.2 Factors that affect UAE ................................................................................ 23
1.4.3 Optimisation of UAE process ...................................................................... 24
CHAPTER 2. MATERIALS AND METHODS .......................................................... 25
2.1 Materials............................................................................................................. 25
2.2 Experimental design ........................................................................................... 25
2.2.1 Screening brown seaweed samples .............................................................. 25
2.2.2 Single factor test .......................................................................................... 25
2.2.3 Response surface methodology (RSM) ........................................................ 26
2.2.4 Preparation of crude extract and fractions .................................................... 27
2.3.1 Total Phenolic Content (TPC) ...................................................................... 29
2.3.2 Antioxidant activities ................................................................................... 30
2.3.3 Tyrosinase inhibitory activity....................................................................... 31
2.3.4 Recovery yield ............................................................................................. 32

2.4 Statistical analysis ........................................................................................... 32
3.1 TPC and antioxidant capacity of five brown seaweed species ............................ 33
3.2 The effect of single factors on the TPC and antioxidant power of Padina
australis extract ........................................................................................................ 35
3.2.1 Influence of ultrasonic temperature .............................................................. 35
3.2.2 Influence of extraction time ......................................................................... 35
3.2.3 Influence of solvent concentration ............................................................... 36
3.2.4 Influence of solid-to-solvent ratio ................................................................ 37
3.3 Modeling of the UAE process ............................................................................ 39
3.4 Effect of extraction variables on experimental responses of TPC, DPPH and
FRAP ………………………………………………………………………………..43
vi


3.5 Optimisation and validation................................................................................ 45
3.6 Recovery yield, TPC, antioxidant and tyrosinase inhibitory activities of Padina
australis crude extract and fractions ......................................................................... 48
CHAPTER 4. CONCLUSION AND RECOMMENDATION .................................... 51
4.1 Conclusion.......................................................................................................... 51
4.2 Recommendation ................................................................................................ 51
REFERENCES ............................................................................................................ 52
APPENDICES ............................................................................................................... I

vii


LIST OF ABBREVIATIONS

UAE


: Ultrasound-assisted Extraction

TPC

: Total Phenol Content

RSM

: Response Surface Methodology

DPPH

: 2,2-Diphenyl-1-picrillhydrazyl

FRAP

: Ferric Reducing Antioxidant Power

GAE

: Gallic Acid Equivalent

TE

: Trolox Equivalent

AAE

: Ascorbic Acid equivalent


DM

: Dry Matter

DD

: Dry Fraction

viii


LIST OF TABLES

Table 1.1. Polysaccharide content of some major brown seaweed .............................. 12
Table 3.1. The TPC and antioxidant capacity of five different brown seaweed species....... 34
Table 3.2. Influence of ultrasound temperature on the TPC and antioxidant capacity of
Padina australis extract ............................................................................................... 36
Table 3.3. Effect of extraction time on the TPC and antioxidant capacity of Padina
australis extract ........................................................................................................... 37
Table 3.4. Effect of solvent concentration on the TPC and antioxidant capacity of
Padina australis extract ............................................................................................... 38
Table 3.5. Effect of solid-to-solvent ratio on the TPC and antioxidant capacity of
Padina australis extract ............................................................................................... 38
Table 3.6. Box-Behnken design and experimental results ........................................... 40
Table 3.7. Analysis of variance for the determination of model adequacy (TPC, DPPH
and FRAP) ................................................................................................................... 43
Table 3.8. Results of regression analysis of experimental values for TPC, DPPH and
FRAP ........................................................................................................................... 44
Table 3.9. Comparisons between two various ratios of sample to solvent for the
selection of optimum UAE conditions ......................................................................... 46

Table 3.10. Recovery yield (% w/w on a dry weight basis), TPC, antioxidant capacity
and tyrosinase inhibitory activity of crude extract and fractions of Padina australis .. 50

ix


LIST OF FIGURES
Figure 1.1. Research conceptual framework .................................................................. 4
Figure 1.2. Structure of some marine algae ................................................................... 6
Figure 1.3. Common applications of seaweed (Sanjeewa & Jeon, 2018)....................... 7
Figure 1.4. Macroscopic appearance of freshwater brown seaweed (Wehr, 2015) ........ 8
Figure 1.5. Chemical structure of fucoxanthin (Abu-Ghannam & Shannon, 2017) ....... 9
Figure 1.6. Chemical structure of alginic acid found in seaweed (Sanjeewa & Jeon, 2018)...... 11
Figure 1.7. Chemical structure of fucoidan (The chain consists of alternating (1-3)- and
(1-4)-linked ɑ-L-fucose residues. R represents attachment of carbohydrate residues
and non-carbohydrate groups) (Venugopal, 2019)....................................................... 11
Figure 1.8. Chemical structures of one unit laminarin (a) M chain (b) G chain (Kadam,
Tiwari et al., 2015) ...................................................................................................... 12
Figure 1.9. Chemical structure of different types of phlorotannins found in seaweed
(Gupta & Abu-Ghannam, 2011) .................................................................................. 14
Figure 1.10. Chemical structure of a typical bromophenol in brown seaweed (Hussain
et al., 2016) .................................................................................................................. 14
Figure 1.11. The pathway for melanin synthesis in mammals (Islam, 2018) ............... 17
Figure 1.12. Enzymatic browning in banana ............................................................... 18
Figure 1.13. Chemical structures of a) L-ascorbic acid b) Kojic acid c) Tropolone d)
Hydroquinone (Chang, 2012) ...................................................................................... 20
Figure 1.14. Schematic representation of an ultrasound equipment (Rojas et al., 2016) ...... 24
Figure 2.1. Flow chart for UAE ................................................................................... 28
Figure 2.2. Brown seaweed samples (a) Sargassum mcclurie (b) Turbinaria ornata (c)
Sargassum duplicatum (d) Sargassum nipponicum (e) Padina australis ..................... 29

Figure 3.1. Actual versus predicted plots for TPC (A), DPPH (B) and FRAP (C) ...... 42

x


Figure 3.2. Prediction profiler showing the effect of X1 (Temperature: 40 – 60oC), X2
(Time: 50 – 80 mins), X3 (Solvent concectration: 0 – 60%), and X4 (Solid-to-Solvent
ratio: 1 – 5 g/100 ml) on the TPC, DPPH and FRAP of Padina australis extract........ 46
Figure 3.3. 3D surface plots and 2D contour plots constructed by JMP software
(version 15) of I –TPC, II – DPPH and III – FRAP of Padina australis extract as a
function of (a) Temperature and Time, (b) Temperature and Solvent concentration, (c)
Temperature and Solid-to-Solvent ratio, (d) Time and Solvent concentration, (e) Time
and Solid-to-Solvent ratio, (f) Solvent concentration and Solid-to-Solvent ratio......... 48

xi


APPENDICES

Appendix 1. Standard curve for total phenol content ..................................................... I
Appendix 2. Standard curve for DPPH radical scavenging activity .............................. I
Appendix 3. Standard curve for FRAP ......................................................................... II
Appendix 4. (a) n-hexane fraction (b) ethyl acetate fraction ........................................ III
Appendix 5. Condensation in a rotavapor .................................................................... III
Appendix 6. Confirmation letter from the editorial office for the submitted manuscript
to the Journal of Food Processing and Preservation (Under review). ..........................IV
Appendix 7. Cover letter of editorial for the submitted manuscript to the Journal of
Food Processing and Preservation (Under review). .....................................................IV

xii



ABSTRACT
The marine environment provides a large, ecologically diverse number of
species that have been found beneficial to human health. Brown seaweeds are an
excellent source of biologically active compounds that can be exploited for their
antioxidant and tyrosinase inhibitory activities. Biologically active compounds from
seaweed have been extracted using different methods, including ultrasound-assisted
extraction, which is favoured due to its low cost and high efficiency.
The present study aimed to determine the optimum ultrasound-assisted
extraction (UAE) conditions for obtaining the highest yield of phenolics and
antioxidants from Vietnamese brown seaweed species. Five brown seaweed species,
Sargassum mcclurei, Turbinaria ornate, Sargassum swartzi, Padina australis and
Sargassum duplicatum, were first screened for their phenolic content and antioxidant
activity. Amongst these seaweeds, Padina australis contained the highest total
phenolic content (TPC) and exhibited the highest ferric reducing antioxidant power
(FRAP). It was thus selected for subsequent studies. Furthermore, single-factor
experiments were carried out to determine the optimal ranges for UAE extraction
parameters temperature, time, solvent concentration and solid-to solvent ratio.
The effects of extraction variables including temperature (40 – 60 C),
extraction time (50 – 80 minutes), solvent concentration (0 – 60%) and solid-tosolvent ratio (1 – 5 g/100 ml) on the total phenolic content (TPC), DPPH radical
scavenging activity and ferric reducing antioxidant power (FRAP) were determined
and optimised using Box-Behnken design in conjunction with Response Surface
Methodology (RSM). The solid-to-solvent ratio was found to be the most important
parameter affecting the yields of the experimental responses. The optimal UAE
conditions were determined to be ultrasonic temperature of 60 C, ultrasonic time of
60 min, solvent concentration of 60% (v/v) aqueous ethanol and solid-to-solvent ratio
of 1 g/100 ml. The predicted values for this condition were validated and found to be
reproducible and repeatable.
The extract obtained at optimised conditions was finally fractionated using

ethyl acetate and n-hexane, and analysed to determine the TPC, antioxidant and
tyrosinase inhibitory activity. The results indicated that the ethyl acetate fraction had
xiii


the highest yields of TPC (807.20 mg GAE/g), DPPH (1417.01 mg TE/g), FRAP
(615.07 mg TE/g) and possessed strong tyrosinase inhibitory activity of 29.90 mg
AAE/g. This establishes that the ethyl acetate fraction of Padina australis may be
further isolated and purified into individual compounds for application as a functional
ingredient in the food and cosmeceutical industry.
Keyword:

Padina

australis,

antioxidant,

ultrasound, phenolics

xiv

optimisation,

seaweed,

tyrosinase,


CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction
Melanin, a dark pigment produced by the skin cells in the innermost layer of the
epidermis, is the primary skin pigment that plays a very crucial role in shielding the
human skin from ultraviolet (UV) radiation (Tu &Tawata, 2015). However, the
excessive accumulation of melanin in the human skin results in hyperpigmentation
disorders such as lentigo, naevus, freckles, age spots, chloasma and melanoma (Kim et
al., 2012; Tu and Tawata, 2015; De Morais et al., 2018). Tyrosinase, a copper enzyme,
is a vital regulator and the rate-limiting enzyme for melanogenesis (Jimenez-cervantes
et al., 1993). It regulates browning reactions in fruits and vegetables. Tyrosinase has
also been implicated in Parkinson’s disease, a progressive neurodegenerative disorder
(Hasegawa, 2010). Therefore, it is crucial to find new natural substances that can
effectively inhibit the enzyme’s activity.
Free radicals and other oxidants are by-products of normal physiological
processes, such as cellular respiration, cell growth regulation and synthesis of
biological substances. They play an essential role in different physiological processes
and have been found to cause a wide range of illnesses (Phaniendra et al., 2015). The
excessive accumulation of free radicals in the body causes oxidative stress. This
phenomenon can lead to several degenerative diseases, such as diabetes,
cardiovascular diseases (CVD), various types of cancer and neurological disorders
(Phaniendra et al., 2015). Antioxidants are compounds that are used to control the
production of these substances. They act by neutralising or capturing oxidative
species, thus minimising oxidative tissue damage (Rodrigues et al., 2019). Generally,
natural antioxidants have considerably low toxicity in addition to being readily
biodegradable, making them an effective substitute for synthetic antioxidants.
Seaweeds are divided into three main classes viz; green (Chlorophytes), brown
(Phaeophytes) and red (Rhodophytes) (Kumar et al., 2008). They have long been
cultivated in Vietnam to be used as food, traditional medicine and more recently,
ingredients for bio-industries. Most brown seaweeds are so-called because they contain
the pigments, fucoxanthin and various pheophycean tannins which mask other pigments,
giving them their characteristic greenish-brown colour (Kumar et al., 2008). Some certain

1


brown seaweeds have been found to possess antihypertensive, antiobesity, anticancer,
antiviral, anti-coagulating and anti-hemorrhagic activities (Iwai, 2008; Zandi et al., 2010;
Soares et al., 2012; Mohamed et al., 2012; Xu et al., 2012).
UAE has proved to be the most economically feasible technology, that is suitable
for the extraction of bioactive compounds due to its low equipment cost, low energy
requirements, low solvent consumption (Chemat et al., 2011; Dang et al., 2017).
Additionally, the use of moderate temperatures makes it suitable for thermolabile
compounds (Bamba et al., 2018). Some factors such as extraction time, ultrasound
temperature, solvent composition, solid-to-solvent ratio, particle size of the raw material,
matrix parameters as well as ultrasonic irradiations (power, frequency) affect the quality,
bioactivity, and composition of extracts obtained by UAE, (Esclapez et al., 2011; da Silva
et al., 2016; Bamba et al., 2018). Recently, some studies have reported the effect of key
ultrasonic factors on antioxidant activity of brown seaweed species. Rodrigues et al.
(2015) determined the impact of UAE on the biological activities of Osmundea pinnatifid
from the central west coast of Portugal. Kadam et al., (2015) investigated the effect of
optimisation of key parameters on the yield of total phenolics, fucose and uronic acid
from Ascophyllum nodosum. Garcia-Vaquero et al., (2018) studied the impact of some
UAE parameters on the yields of fucose, total glucans and antioxidant activities from
Laminaria digitata. Dang et al., (2017) optimised UAE for maximum antioxidant activities
and total phenolic content (TPC) of the H. banksii. No study has however been conducted
on the effect of key UAE parameters on the total phenol content and antioxidant activity
of Vietnamese brown seaweed species, particularly Padina australis. Therefore, this study
aimed to investigate the effect of UAE variables (ultrasound temperature, extraction time,
solvent concentration and solid-to-solvent ratio) on the total phenol content and
antioxidant activities and to optimise the UAE variables to obtain high yields of phenolic
compounds, antioxidant and tyrosinase inhibitory activities of Padina australis.
1.1.1 Problem statement

The overproduction of melanin in different specific parts of the skin can result
in hyper-pigmentary disorders, and aesthetic problems in humans (Chang, 2009).
Melanosis, one of the most common forms of acquired hyperpigmentation, is
characterised by irregular brown patches on sun-exposed skin. It often affects the
populations with darker skin complexion, with greater severity and higher frequency in
2


Hispanics and Asians (Stratigos & Katsambas, 2004). In these populations, there is an
increasing demand for skin whitening agents and other related cosmetics product.
Several tyrosinase inhibitors have been reported from natural sources, but only
a few of them are used as skin-whitening agents, primarily due to various safety
concerns. For example, linoleic acid, hinokitiol, kojic acid, arbutin, naturally occurring
hydroquinones, and catechols were reported to inhibit enzyme activity but also
exhibited side effects. Therefore there is the constant search for cosmetic products that
are not only safe but contain biologically active ingredients that possess medical or
drug-like benefits in addition to their skin whitening effect.
1.1.2 Research questions
This research seeks to answer the following questions:
Does Vietnamese brown seaweed contain phenolics and possess antioxidant
and tyrosinase inhibitory activities?
What UAE optimisation condition is best for maximum yield of phenolics and
antioxidant activity?
1.1.3 Hypothesis
Brown seaweed species may possess tyrosinase inhibitory activity
Brown seaweed species extracted by UAE may possess high phenolics,
antioxidant and tyrosinase inhibitory activities.
1.1.4 Prediction
Extraction of Vietnamese brown seaweed bioactive compounds using UAE will
improve the tyrosinase inhibitory activity, thus making it suitable for use in the

cosmeceutical and food industry.
1.1.5 Main objectives
The main objective of this study is to investigate the phenolic content,
antioxidant and tyrosinase inhibitory activities of some brown seaweed species
harvested in Vietnam.

3


1.1.6 Specific objectives
 To screen five Vietnamese brown seaweed species based on their phenolics and
antioxidant activity in order to select the starting material.
 To optimise key UAE parameters to obtain high yields of phenolics and
antioxidant activity from selected brown seaweed.
 To fractionate crude extract of selected brown seaweed obtained from
optimised UAE conditions with different solvents.
1.1.7 Conceptual framework
SCREENING
Conventional extraction

TPC, DPPH, FRAP

SINGLE FACTOR EXPERIMENT
UAE (Time, temperature, solvent concentration,
solid-to-solvent ration)

TPC, DPPH, FRAP

OPTIMISATION (RSM)
UAE (Time, temperature, solvent concentration,

solid-to-solvent ration)

TPC, DPPH, FRAP

FRACTIONATION
solvents (ethyl acetate, n-hexane, water)

Tyrosinase inhibitory activity, TPC, DPPH, FRAP.

Figure 1.1. Research conceptual framework
1.2. Literature review
1.2.1 Seaweed
The marine environment covers over 70% of the earth’s surface and provides a
broad range of highly ecological, chemical, and, biological diversity starting from
microorganisms to vertebrates (Karupanan & Sutlana, 2017). One of these organisms
is marine algae, one of the largest producers of important marine living, renewable
4


resources (Saranraj et al., 2014). Marine algae are a diverse group of oxygengenerating, chlorophyll-containing, photosynthetic organisms which inhabit the marine
environment and are classified as either microalgae or macroalgae. Microalgae are
small (µM) unicellular organisms while macroalgae, on the other hand, are large,
multicellular organisms.
Seaweeds are macroscopic algae found freely floating or attached to the bottom
of rocks, dead corals, pebbles, shells and, other plant materials (Rindi et al., 2012;
Pati et al., 2016). They are so-called because of their abundance in seas and oceans.
They are found in relatively shallow coastal waters, estuaries, intertidal and deep-sea
areas up to 180 meters depth (Pati et al., 2016). They are primitive plants with little
tissue differentiation, no roots, no true vascular tissues, stems or leaves, and flowers
(Chia, 2010; Zandi et al., 2010). They belong to the division of Thallophyta in the

plant kingdom. They are not only frequently classified based on their photosynthetic
pigments but also by differences in many ultra-structural and biochemical features
including the type of storage material, cell wall composition, presence/absence of
flagella, and the fine structure of the chloroplasts (Rindi et al., 2012; Mekinić et al.,
2019). Based on these features, marine algae are broadly classified into four groups
namely Chlorophyceae (green algae), Phaeophyceae (brown algae), Rhodophyceae
(red algae), and, Cyanophyceae (blue-green algae) (Lavanya et al., 2017). The group
brown, green, and, red algae consist of approximately 1,800 1,200 and 6000 species,
respectively.
Seaweeds contain a large variety of beneficial compounds, and for this reason,
they have been used in many parts of the world as a source of essential nutrients and
ingredient in cosmeceutical and industrial applications. They are also known to be an
extremely rich source of biologically active substances with health-promoting ability
(Wijesinghe & Jeon, 2011). Their biological activities are correlated to the presence of
secondary metabolites, present as the plants’ defence mechanism against extreme
marine environmental conditions. These bioactive compounds include soluble
polysaccharides, sulfated polysaccharides, carotenoids, omega-3 fatty acids, vitamins,
tocopherols, and, phycocyanins and have been extracted using different extraction
techniques. Figure 1.2 shows the structure of some seaweed.

5


Figure 1.2. Structure of some marine algae
1.2.2 Seaweed in Vietnam
Vietnam’s coastline is estimated to be around 3260 km with her climate varying
from subtropical in the northern part which gradually becomes tropical in the southern
part of the country (Hong et al., 2007). These physical and climatological
characteristics are suitable for the cultivation of seaweed. Vietnam has an abundance
of algae floral with the total number of species estimated to be nearly 1000 spp with

about 827 species already identified (Tu et al., 2013). The traditional Vietnamese
coastal people have harvested and utilised seaweeds for over one hundred years.
However, the use of seaweeds is limited to people living in coastal areas (Hong et al.,
2007). Seaweeds play a vital role as a source of food and ingredients in traditional
Vietnamese medicine. Vietnamese consume seaweed as fresh vegetables, salad, soups,
or snacks. Apart from food uses, seaweeds are widely used as ingredients in various
industries such as cosmetology, pharmaceutical, animal feed, and fertiliser industries.
The schematic representation of the common application of seaweed in Vietnam is
shown in Figure 1.3.

6


Large scale commercial cultivation for

Harvesting in the wild for industrial

industrial and traditional applications

and traditional applications

Food

Medicine/pharmacy

Industry

Figure 1.3. Common applications of seaweed (Sanjeewa & Jeon, 2018)
1.2.3 Brown seaweed
Brown algae (Phaeophyceae) have received interest from researchers due to

their large variety of bioactive compounds and nutritional value. The brown algae are
described as the largest and most diverse class of macroalgae. They range from small
filamentous forms to large, complex seaweed (Rindi et al., 2012). Most brown
seaweeds are so-called because they contain the xanthophyll pigment viz., fucoxanthin
and various pheophycean tannins which mask other pigments such as chlorophyll a
and chlorophyll b, thus giving them their characteristic greenish-brown colour (Kumar
et al., 2008).
All members of the phaeophyceae are multicellular, none are unicellular in the
vegetative phase, this is unlike other groups in the same phyllum (Wehr, 2015). Most
species have an alternation of haploid and diploid generations, which may be either
isomorphic or heteromorphic (Wehr, 2015). Many are macroscopic seaweeds with
complex tissues and reproductive structures, although many simpler filamentous forms
exist. There are approximately 1,800 species in this class of macroalgae.
7


Figure 1.4. Macroscopic appearance of freshwater brown seaweed (Wehr, 2015)
1.2.4 Major antioxidant constituents of brown seaweed
Oxidative stress in humans has been found to be the result of an imbalance
between the homeostasis of prooxidants and antioxidants thus leading to the generation of
free radicals and other toxic reactive oxygen or nitrogen species (ROS and RNS)
(Vadlapudi et al., 2012). Researches done over the past two decades have demonstrated
that oxidative stress is involved in the pathogenesis and pathophysiology of many chronic
diseases and inflammation (Blomhoff et al., 2006). Antioxidants, also known as free
radical scavengers, in simplest terms are defined as redox-active compounds that can
reduce oxidative stress by reacting non-enzymatically with free radicals and reactive
oxygen or nitrogen species thus helping the body maintain normal physiological balance.
Epidemiological evidence has shown that high antioxidant activity in plant materials are
highly correlated with their potential health benefits.
Studies of the antioxidant constituents of brown seaweed have been conducted

over the years and have been primarily classified as pigments, polysaccharides,
polyphenols, vitamins, and enzymes (Holdt & Kraan, 2011). The bioactive compounds
all exhibit some degree of antioxidant, prooxidant or synergistic activity. The
following section will discuss the properties of fucoidans, carotenoids, polyphenolic
compounds and enzymes found in brown seaweed and their influence on the
antioxidant efficacy of brown seaweed extracts.
8


Pigments
Brown seaweed contains a diverse number of photosynthetic pigments such as
carotenoids and xanthophyll, which are responsible for its characteristic golden-brown
colour and serve as an important source of antioxidant. Fucoxanthin, a xanthophyll, found
in the chloroplast of algal cells is the primary biologically active pigment present in brown
seaweed. It accounts for more than 10% of the estimated total production of carotenoids in
the marine environment (Peng et al., 2011). Fucoxanthin, shown in Figure 1.5, has been
found to act as an antioxidant under anoxic conditions, i.e. in the presence of meagre
amounts of oxygen typical of most animal tissues under normal physiologic conditions,
thus making it a potent free radical scavenger (D’Orazio et al., 2012). Industrially
Undaria pinnatifida is the most widely utilised brown seaweed for fucoxanthin extraction
of because of the high concentration of the pigment in its lipid extract (Abu-Ghannam &
Shannon, 2017). It has also been extracted and isolated from other species like Undaria
pinnatifida, Hijikia fusiformis, Laminaria japonica, Undaria pinnatifida, and, Sargassum
fulvellum ( Roh et al., 2008; D’Orazio et al., 2012;). Studies have shown that brown algae
pigments possess anti-cancer, anti-obesity, and, anti-diabetic properties which have been
found to be mainly because of their antioxidant capacity (Gammone & D’Orazio, 2015;
Usoltseva et al., 2018).

Figure 1.5. Chemical structure of fucoxanthin (Abu-Ghannam & Shannon, 2017)
Polysaccharide

Seaweeds contain a significant amount of soluble polysaccharides and have the
potential function as dietary fibre. They are the main component of seaweeds,
constituting about 70% of their dry weight (Holdt & Kraan, 2011). The polysaccharide
content of some major brown seaweed is presented on Table 1.1. Biologically,
polysaccharides function to provide structure to the plants and physically support their
thallus in water (Venugopal, 2019). They also act as antioxidants thus protecting the
9


plant

from

oxidative

stress

Polysaccharides

consist

of

several

units

of

monosaccharides linked together by glycosidic bonds and they usually differ from one

another by the number of glycosidic bonds, type of functional group, type of
branching, molecular weight, and, chain length. In a broad sense, algae
polysaccharides are classified as sulfated and non-sulfated. Sulfated polysaccharides
include fucoidans, agar and carrageenans and ulvans, while the most commercially
important non-sulfated polysaccharide is alginate. Fucoidan, laminarin, and, alginic
acid are the significant polysaccharides found in brown seaweed (Okolie et al., 2017).
Fucoidans
They are a group of fucose-rich, sulfated polysaccharides, consisting of αlinked l-fucose residues having various substitutions (Ale & Meyer, 2013). They are
found in the cell wall of cell walls of brown seaweeds including Sargassum spp. and
Fucus vesiculosus (Venugopal, 2019). They are water-soluble and are constituted
mainly of sulfated α-L-fucopyranose residues and may contain monosaccharides such
as glucose, mannose, galactose, xylose and some acetylated groups (Kumar et al.,
2008). The chemical structure of fucoidan shown in Figure 1.7, differ according to the
seaweed species, but they are mainly composed of fucose and sulphates.
Laminarin
Laminarins, also called laminarans, are the major food reserve of brown algae.
They are a class of low-molecular-weight storage β-glucans, composed of (1,3)-β-Dglucan (Kadam et al., 2015). There are two types of laminarin chains depending on the
reducing end; they are M and G chains. The M chains end with 1-O-substituted Dmannitol, whereas G chains end with glucose as the reducing end (Kadam, Tiwari, et
al., 2015). Laminarins can be either soluble or insoluble depending on the degree of
branching. They form complex structures that are stabilised by inter-chain hydrogen
bonds and are therefore resistant to digestion in the upper gastrointestinal tract (GIT)
making them a good source of dietary fibres (Gupta & Abu-Ghannam, 2011).
Laminarin has been isolated and purified from seaweeds Saccharina latissima,
Laminaria digitata, Laminaria hyperborea, Fucus vesiculosus, and, Ascophyllum
nodosum (Kadam et al., 2014; Sterner & Edlund, 2016). The structures of M and G
chain types of laminarin are shown in Figure 1.8.

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Alginic acid
Also called alginate or algin, is an alkali-soluble polysaccharide consisting of 1,
4-linked b-D-mannuronic and a-L-guluronic acid residues arranged irregularly along the
chain (Wijesinghe & Jeon, 2011). It is the main structural polysaccharide in brown algae
and is found distributed widely in the cell walls and intracellular materials. Alginates
produced by brown seaweed are commonly used in the food and pharmaceutical
industries due to their ability to chelate metal ions and form highly viscous solutions
(Gupta & Abu-Ghannam, 2011). In addition to this, they possess potent antioxidant
activity, the chemical structure of alginic acid is shown in Figure 1.6.

Figure 1.6. Chemical structure of alginic acid found in seaweed (Sanjeewa & Jeon, 2018)

Figure 1.7. Chemical structure of fucoidan (The chain consists of alternating (1-3)and (1-4)-linked ɑ-L-fucose residues. R represents attachment of carbohydrate
residues and non-carbohydrate groups) (Venugopal, 2019)

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