Brown algae as a source of bioactive compounds for pancreatic cancer treatment

Fucoxanthin content, isolation and cytotoxic activity against pancreatic cancer from brown alga Hormosira banksii Turner Decaisne.. Van Altena as co-authors with contribution of planning

compounds for pancreatic cancer treatment

Thanh Trung Dang

B.Eng (Nha Trang University, Khanh Hoa, Vietnam)

MSc (Nha Trang University, Khanh Hoa, Vietnam)

A thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy in Food Science

School of Environmental and Life Sciences, Faculty of Science

University of Newcastle Australia

May 2018

STATEMENT OF ORIGINALITY

I hereby certify that to the best of my knowledge and belief this thesis is my own work and contains no material previously published or written by another person except where due references and acknowledgements are made It contains no material which has been previously submitted by me for the award of any other degree or diploma in any university or other tertiary institution.

Thanh Trung Dang

Date: 6/5/2018

DECLARATION OF AUTHORSHIP

I hereby certify that this thesis is in the form of a series of 8 papers I have included as part of

the thesis a written statement from each co-author, endorsed in writing by the Faculty Assistant Dean (Research Training), attesting to my contribution to any jointly authored papers.

Thanh Trung Dang

Date: 6/5/2018

ACKNOWLEDGEMENTS

Firstly, I would like to give a great appreciation to my supervisors: Principal supervisor: A/Prof Christopher J Scarlett; Co-supervisors: A/Prof Michael C Bowyer and Dr Ian A Van Altena for their supervision and support during my PhD course The suggestions and encouragement from the supervisor panel played an important role in my research achievements

I acknowledge the financial support from University of Newcastle; the Vietnamese Government through the Ministry of Education and Training; the Ministry of Agriculture and Rural Development for awarding a VIED-TUIT scholarship, which enabled me to study for a PhD at the University of Newcastle, with full cover for academic expenses, as well as living and travellingallowances

I highly appreciated the contribution of brown algae as the material for my PhD project from

Dr Maria Schreider (School of Environmental and Life Sciences, Faculty of Science, University of Newcastle), in particular for identifying algal species and allowing her students assist in the collection of the samples

I would like to say thank you to Dr Quan V Vuong, Dr Danielle Bond and other PhD students

in Food Science, technical staff and administrative staff for helping me to overcome the difficulties in the laboratory and administrative works

Finally, I would like to say thank my colleagues and friends from Nha Trang University, Vietnam for their encouragement A very special thank you is given to my family (parents, young brother), who have always been behind me, encouraging and inspiring me during my PhD project

LIST OF PUBLICATIONS INCLUDED AS PART OF THE THESIS

I warrant that I have obtained, where necessary, permission from the copyright owners to use any third party copyright material reproduced in the thesis, or to use any of my own published work in which the copyright is held by another party

1 Paper I: Dang TT, Vuong QV, Schreider MJ, Bowyer MC, Van Altena IA & Scarlett CJ

Brown Alga (Hormosira banksii (Turner) Decaisne) J Food Process Pres 41(4): e13025

doi.org/10.1111/jfpp.13025

2 Paper II: Dang TT, Bowyer MC, Van Altena IA & Scarlett CJ (2018) Comparison of

chemical profile and antioxidant properties of the brown algae, Inter Food Sci Technol 51(1):

174-181 doi: 10.1111/ijfs.13571

3 Paper III: Dang TT, Vuong QV, Schreider MJ, Bowyer MC, Van Altena IA & Scarlett

CJ (2017) Optimisation of ultrasound-assisted extraction conditions for phenolic content and

antioxidant activities of the alga Hormosira banksii using response surface methodology J

Appl Phycol 29(6): 3161-3173 doi.org/10.1007/s10811-017-1162-y

4 Paper IV: Dang TT, Bowyer MC, Van Altena IA & Scarlett CJ (2017) Optimum

conditions of microwave assisted extraction for phenolic compounds and antioxidant capacity

doi.org/10.1080/01496395.2017.1414845

5 Paper V: Dang TT, Vuong QV, Bowyer MC & Scarlett CJ Chemical profile and

antioxidant activities of the crude extract and different fractions prepared from the brown alga

Hormosira banksii (Turner) Decaisne Submitted to Journal of Botanica Marina

6 Paper VI: Dang TT, Bhuyan DJ, Bond DR, Bowyer MC, Van Altena IA & Scarlett CJ

Fucoxanthin content, isolation and cytotoxic activity against pancreatic cancer from brown alga

Hormosira banksii (Turner) Decaisne Submitted to Journal of Biotechnology

7 Paper VII: Dang TT, Sakoff JA, Bowyer MC, Van Altena IA & Scarlett CJ Antioxidant

and cytotoxic activity (in vitro) of phlorotannin-enriched fractions from the brown alga

Hormosira banksii (Turner) Decaisne Submitted to Journal of MarineBiotechnology

8 Paper VIII: Dang TT & Scarlett CJ Extraction and cytotoxic activity of the sulfated

polysaccharides (fucoidans) against pancreatic cancer in vitro from brown alga Hormosira

banksii (Turner) Decaisne Submitted to Journal of Biomedicine andPharmacotherapy

STATEMENT OF AUTHORS' CONTRIBUTION TO THE PAPERS

To whom it may concern,

We, Quan V Vuong, Maria J Schreider, Christopher J Scarlett, Michael C Bowyer and Ian A Van Altena as co-authors with contribution of planning, giving the suggestions and editing the paper, attest that research higher degree candidate, Thanh Trung Dang, was the principle contributor to the planning, execution, analyses of the experiments and the writing of the published research paper entitled “The Effects of Drying on Physico‐Chemical

Properties and Antioxidant Capacity of the Brown Alga (Hormosira banksii (Turner) Decaisne)” J Food Process Pres 41(4): e13025 doi.org/10.1111/jfpp.13025

Dr Ian A Van Altena

Professor Frances Martin

Assistant Dean Research Training (ADRT)

Date:

STATEMENT OF AUTHORS' CONTRIBUTION TO THE PAPERS

To whom it may concern,

We, Christopher J Scarlett, Michael C Bowyer and Ian A Van Altena as co-authors with contribution of planning, giving the giving the suggestions and editing the paper, attest that research higher degree candidate, Thanh Trung Dang, was the principle contributor to the planning, execution, analyses of the experiments and the writing of the published research paper entitled “Comparisonof chemical profileandantioxidantproperties of the brown algae”,

Inter Food Sci Technol 51(1): 174-181 doi:10.1111/ijfs.13571

Dr Ian A Van Altena

Professor Frances Martin

Assistant Dean Research Training (ADRT)

Date:

STATEMENT OF AUTHORS' CONTRIBUTION TO THE PAPERS

To whom it may concern,

We, Quan V Vuong, Maria J Schreider, Christopher J Scarlett, Michael C Bowyer and Ian A Van Altena as co-authors with contribution of planning, giving the giving the suggestions and editing the paper, attest that research higher degree candidate, Thanh Trung Dang, was the principle contributor to the planning, execution, analyses of the experiments and the writing of the published research paper entitled “Optimisation of ultrasound-assisted extraction conditions for phenolic content and antioxidant activities of the

alga Hormosira banksii using response surface methodology” J Appl Phycol 29(6): 3161-

I have seen this paper and I agree with the signatories above that it represents a substantial

amount of research work and can be part of Thanh Trung Dang’ s PhD thesis

Professor Frances Martin

Assistant Dean Research Training (ADRT)

Date:

STATEMENT OF AUTHORS' CONTRIBUTION TO THE PAPERS

To whom it may concern,

We, Christopher J Scarlett, Michael C Bowyer and Ian A Van Altena as co-authors with contribution of planning, giving the feedbacks and editing the paper, attest that research higher degree candidate, Thanh Trung Dang, was the principle contributor to the planning, execution, analyses of the experiments and the writing of the published research paper entitled “Optimum conditions of microwave assisted extraction for phenolic compounds and antioxidant capacity

I have seen this paper and I agree with the signatories above that it represents a substantial

amount of research work and can be part of Thanh Trung Dang’ s PhD thesis

Professor Frances Martin

Assistant Dean Research Training (ADRT)

Date:

To whom it may concern,

We, Quan V Vuong, Christopher J Scarlett, Michael C Bowyer as co-authors with contribution of planning, giving the suggestions and editing the paper, attest that research higher degree candidate, Thanh Trung Dang, was the principle contributor to the planning, execution, analyses of the experiments and the writing of the published research paper entitled “Chemical profile and antioxidant activities of the crude extract and different fractions

prepared from the brown alga Hormosira banksii (Turner) Decaisne” Submitted to Journal of

Botanica Marina

Thanh Trung Dang

Date: 26/4/2018

I have seen this paper and I agree with the signatories above that it represents a substantial amount

of research work and can be part of Thanh Trung Dang’ s PhD thesis

Professor Frances Martin

Assistant Dean Research Training (ADRT)

Date:

To whom it may concern,

A Van Altena as co-authors with contribution of planning, giving the giving the suggestions and editing the paper, attest that research higher degree candidate, Thanh Trung Dang, was the principle contributor to the planning, execution, analyses of the experiments and the writing of the published research paper entitled “Fucoxanthin content,

isolation and cytotoxic activity against pancreatic cancer from brown alga Hormosira banksii

(Turner) Decaisne” Submitted to Journal Biotechnology

Dr Ian A Van Altena

Date: 27/4/2018

Thanh Trung Dang

Date: 26/4/2018

I have seen this paper and I agree with the signatories above that it represents a substantial amount

of research work and can be part of Thanh Trung Dang’ s PhD thesis

Professor Frances Martin

Assistant Dean Research Training (ADRT)

Date:

To whom it may concern,

We, Christopher J Scarlett, Jennette Sakoff, Michael C Bowyer and Ian A Van Altena as co-authors with contribution of planning, giving the suggestions and editing the paper and Jennette Sakoff (conducting experiments of cancer cells), attest that research higher degree candidate, Thanh Trung Dang, was the principle contributor to the planning, execution, analyses of the experiments and the writing of the research paper entitled “Antioxidant and

cytotoxic activity (in vitro) of phlorotannin-enriched fractions from the brown alga Hormosira

banksii (Turner) Decaisne” Submitted to Journal of Marine Biotechnology

Dr Ian A Van Altena

Date: 27/4/2018

Thanh Trung Dang

Date: 26/4/2018

I have seen this paper and I agree with the signatories above that it represents a substantial

amount of research work and can be part of Thanh Trung Dang’ s PhD thesis

Professor Frances Martin

Assistant Dean Research Training (ADRT)

Date:

To whom it may concern,

Christopher J Scarlett as co-authors with contribution of planning, giving the suggestions and editing the paper, attest that research higher degree candidate, Thanh Trung Dang, was the principle contributor to the planning, execution, analyses of the experiments and the writing of the research paper entitled “Extraction and cytotoxic activity of the sulfated

polysaccharides (fucoidans) against pancreatic cancer in vitro from brown alga Hormosira

banksii (Turner) Decaisne Submitted to Journal of Biomedicine and Pharmacotherapy

Thanh Trung Dang

Date: 26/4/2018

I have seen this paper and I agree with the signatories above that it represents a substantial

amount of research work and can be part of Thanh Trung Dang’ s PhD thesis

Professor Frances Martin

Assistant Dean Research Training (ADRT)

Date:

CONFERENCE PRESENTATIONS

1 Thanh T Dang, Quan V Vuong, Micheal C Bowyer, Ian A Van Altena, Christopher J

Scarlett Phytochemical and antioxidant properties of crude and fractionated extracts from

the brown alga Hormosira banksii (Turner) Decaisne International scientific conference

“Sustainable Agriculture and Environment” December, 13-14, 2016, Ho Chi Minh City,

Vietnam Oral presentation

2 Thanh T Dang, Quan V Vuong, Maria J Schreider, Micheal C Bowyer, Ian A Van

Altena, Christopher J Scarlett Effect of drying methods on chemical properties and

Sustainable Agriculture, Food and Energy November, 17-20, 2015, Ho Chi Minh City,

Vietnam Oral presentation

Figure 1: Stran-fucoxanthin

Figure 2: Some phlorotannins with phloroglucinol (1,3,5-trihydroxybenzene) as a basic unit

Figure 2: Some phlorotannins with phloroglucinol as a basic unit (continue)

Figure 3: Sulfated polysaccharides (Fucoidans)

Structure of fucoidan unit

STATEMENT OF ORIGINALITY - i

DECLATATION OF AUTHORSHIP - ii

ACKNOWLEDGEMENTS - iii

LIST OF PUBLICATIONS INCLUDED AS PART OFTHE THESIS - iv

STATEMENTS OF AUTHOR S’ CONTRIBUTION TOTHE PAPERS - vi

CONFERENCE PRESENTATIONS - xiv

LIST OF ABBREVIATIONS - xv

FIGURES FOR THE THESIS - xvi

TABLE OF CONTENTS - xviii

ABSTRACT - xx

PART 1: LITERATURE REVIEW 1 1.1 Background 1 1.1.1 Algae and health benefits 1 a) Overview of marine algae 1 b) Health benefits 2 1.1.2 Bioactive compounds from brown algae 4 a) Fucoxanthin 4 b) Pholorotannins 5 c) Polysaccharides (fucoidans) 6 d) Other components 7 1.1.3 Extraction of algal compounds 8 a) Microwave Assisted Extraction 9 b) Ultrasound Assisted Extraction 10 1.1.4 Isolation and identification of bioactive compounds 10 a) Chromatographic separation and isolation ofbioactive compounds 10 b) Identification of bioactive compounds 15 1.1.5 Activities of algal compounds against several cancer cell lines 16 1.1.6 Algal components against pancreatic cancer cell lines 18 a) Problems with pancreatic cancer 18 b) Algal compounds against pancreatic cancer 19 1.2 Research content 20 1.3 Research Aims and Expected Outcomes 21 1.4 Experimental Rationale 21 1.5 Hypothesis, aims and objectives 23 PART 2: RESULTS 26 2.1 Synopsis of research papers published from results 26

3.1 General discussion 34

Marine macro-algae (seaweeds) are a rich source of bioactive compounds that have the potential to be used as functional constituents for human health applications Bioactive compounds from brown algae include pigments, sulfated polysaccharides (fucoidans), phlorotannins (marine phenolics), terpenes and other secondary metabolites These compounds have been reported to possess biological activity against a range of chronic diseases, including cancer

Pancreatic cancer has a high mortality rate and short survival timeline due to difficulties associated with achieving a correct diagnosis at an early stage of the disease, a predisposition

to metastasise to other organs within the body, and a lack of progress in the development of new therapeutic strategies For decades, gemcitabine has remained the single front-line chemotherapeutic agent for treating advanced adenocarcinoma of the pancreas A low proportion of patients however see direct and meaningful benefit from gemcitabine, with current combinatorial chemo-radiation treatment regimens delivering only limited survival benefits

Brown algae Sargassum vestitum; Sargassum linearifolium; Phyllospora comosa; Padina sp.;

Hormosira banksii and Sargassum podocanthum are species found in Eastern Coast of NSW,

Australia Despite their relative abundance, knowledge of the phytochemical properties of extracts from these species remains limited Therefore, an assessment of the bioactive potential

of compounds derived from these brown algae against pancreatic cancer is justified

The current study hypothesised that the physico-chemical profile and antioxidant activities of bioactive compounds derived from brown algae could be improved through optimisation of drying and extraction conditions, and that phytochemical fractions or individual compounds isolated from the algae display efficacy against pancreatic cancer cell lines

• Optimise drying conditions for algae to preserve chemical activity and antioxidant

power

• Optimise the extraction of phenolics from algae using irradiative technologies such as ultrasound and microwave

• Isolate key components including fucoxanthin, phenolics and polysaccharides for

assessment of their cytotoxic activity against a range of pancreatic cancer celllines

Six drying methods were applied to H banksii samples including de-humidification, vacuum

and freeze drying, sun drying (in direct sunlight), as well as microwave and oven drying The

drying, while sun drying produced the highest bioactive compound yield and was the most cost effective

Among the six algal extracts, H banksii possessed the highest total phenolic content (TPC)

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content was present in all six extracts, with four species - Padina sp.; S linearifolium; S

vestitum and S podocanthum possessing with high concentrations (1.97; 1.76; 1.65 and 1.46

mg fucoxanthin.g-1) respectively

Extraction of phenolics from H banksii using aqueous ethanol (70%) and ultrasonic irradiation

improved both total yield and antioxidant activity Optimal extraction conditions were

(150w) The highest values of TPC and antioxidant activity (ABTS, DPPH and FRAP) achieved were 23.12 mg GAE.g-1, 85.64 TE.g-1, 47.24 TE.g-1 and 12.56 TE.g-1, respectively

ethanol 70% as a solvent From the preliminary experiments and literature, acetone was proved

to be the best solvent in relation to yields of phenolics and produced the highest antioxidant activity compared to methanol, water and ethyl acetate However, ethanol was preferred on safety grounds for usage as well as minimal solvent residue in the sample Therefore, ethanol

70% is suitable for extraction of phenolics from S vestitum The maximal values of TPC and

antioxidant activities gained from this alga were 58.2 mg GAE.g-1, 149.84 TE.g-1, 116.54 TE.g-

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and 67.95 TE.g-1, respectively with the optimal conditions determined to be irradiation time

of 75 seconds, ethanol percentage of 70% and power of 80% (1080w)

Polysaccharides from H.banksii were extracted using water and separated by precipitation

using pure ethanol and centrifugation Sulfated polysaccharides (fucoidans) were observed in three fractions (CF50, CF70 and CFR) Antioxidant activities of the CF50 and CF70 fractions were low, while high activity was observed in the CFR fraction due to the presence of high concentrations of phenolics

Finally, from the H.banksii extract, fucoxanthin was isolated through solvent partitioning and

column chromatography techniques, in high purity (92.3%; validated by HPLC) Phenolic compounds were separated in solvent fractions of differing polarity (hexane, dichloromethane, ethyl acetate and butanol fractions), with ethyl acetate possessing the highest TPC value and antioxidant activities Sulfated polysaccharides (fucoidan) with higher sulfate content were found in the CF70 fraction Fucoxanthin, phenolic and polysaccharide fractions were

investigated for cytotoxic activity against a range of pancreatic cancer cell lines With a range

acetate fraction) showed excellent cancer cell growth inhibition properties (70-100%) against Mia PaCa-2, BxPC-3 and CFPAC-1 cell lines but was also toxic towards normal pancreas cells

xxii

Fucoxanthin was also a potent agent against pancreatic cancer cell lines with high growth

fractions (CF50 and CF70) showed quite high activity (inhibition of 39.35 – 82.82% at the concentrations of 100–200 µg.mL-1) against these cancer cells with low toxicity towards normal cells (IC50 values were 526.32 µg.mL-1 (CF50) and 781.25 µg.mL-1 (CF70))

In summary, the hypothesis was supported and the aims were achieved in these studies Three out of six drying methods were found to be effective for producing higher yields of phenolics and stronger antioxidant activities The bioactive components extracted using ultrasound and microwave techniques were optimised for high efficacy of phenolics and antioxidant activities

In addition, purified fucoxanthin and polar phenolics obtained by partition and column

chromatography showed strong potential against pancreatic cancer cell lines Sulfated

polysaccharide fractions (CF50 and CF70) exhibited high cytotoxic effects against pancreatic cancer cell lines with less toxicity to non-tumorigenic cells These algal components have potential application in the functional food and pharmaceutical industries

1

1.1 Background

1.1.1 Algae and health benefits

a) Overview of marine algae

Marine algae are one of the largest biomass producers in the marine environment Algae, by definition, do not possess true roots or stems They are found in all corners of the globe and occur in a range of sizes and morphologies Algae are classified into two major sub-groups based on size; macro-algae and micro-algae (Bocanegra et al., 2009) Micro-algae are microscopic organisms and include blue-green algae (Cyanobateria), diatoms (Bacillariophyta) and dinoflagellates (Dinophyceae) (Garson, 1989) Macro-algae (seaweeds) are divided into three types; brown algae (Phaeophyceae), red algae (Rhodophyceae) and green algae (Chlorophyceae) In macro-algae, pigments and their phytochemical profile are the most common features used in algal classification In brown algae, fucoxanthin is the major pigment, while the predominant polysaccharides including

alginates, laminarins, fucans and celluloses In green algae, chlorophyll a and b are the

dominant pigments, with ulvan being the major polysaccharide component, while coloration

in red algae is derived from the presence of two pigment-protein complexes-phycoerythrin

and phycocyanin (Bocanegra et al., 2009; O’Sullivan et al., 2010)

Macro-algae are a source of biologically active phytochemicals including carotenoids, polyphenols, polysaccharides, polyunsaturated fatty acids and lipids They are also an excellent source of vitamins such as A, B1, B12, C, D and E, riboflavin, niacin, pantothenic acid and folic acid, as well as minerals such as Ca, P, Na, K (Gupta & Abu-Ghannam, 2011) These compounds are known to possess biological activities and hence have potential

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nutritional compositions of macro-algae are affected by a number of factors including algal species, stage of development, habitat, seasonality, and the local environment (Bocanegra et al., 2009; Lordan et al., 2011; Mohamed et al., 2012)

b) Health benefits

Asian countries, including Korea, Japan, and China, consume the greatest proportion of algae per capita annually, while in Western countries, the majority of algae is utilised in industrial applications (Brown et al., 2014) Correlations between algae consumption and health benefits, including digestive health and weight management and lower incidence of chronic diseases such as cancer, hyperlipidaemia and coronary heart disease, have been well documented (Brown et al., 2014; Gupta & Abu-Ghannam, 2011)

Consumption of fucoxanthin (present in brown algae) in combination with fish oils has been linked to increased metabolism, weight control and reduced blood glucose in obese/diabetic

fat tissue, a process also known as thermogenesis (Okada et al., 2011), which reduces cardiovascular damage caused by associated risk factors such as obesity, diabetes, high blood pressure, chronic inflammation, plasma and hepatic triglyceride, and cholesterol concentration (Jeon et al., 2010) The health benefits of fucoxanthin also include antioxidant and anticancer activity Anticancer activity occurs via several different mechanisms, including anti-proliferation, induction of apoptosis, cell cycle arrest and anti-angiogenesis (Rengarajan et al., 2013) Toxicity studies in mice showed fucoxanthin to be safe to consume, with no abnormal changes in liver, kidney, spleen and gonadal tissues reported (Beppu et al., 2009)

Marine phenolics (phlorotannins), the natural antioxidants mainly found in edible brown algae, can protect food products against oxidative degradation as well as preventing and/or

3

have been demonstrated by a number of studies including antioxidant (Ahn et al., 2007), antimicrobial (Eom et al., 2012), anti-allergic (Sugiura et al., 2007), anti-diabetic (Seung-Hong Lee & Jeon, 2013; Nwosu et al., 2011), anti-HIV (Thomas & Kim, 2011) and anti-cancer (Dellai et al., 2013; Li et al., 2011) activities

for the digestion of oligosaccharides, glucose absorption and the maintenance of glucose levels in plasma, leading to the suppression of postprandial hyperglycemia) (Thomas & Kim, 2011), and Angiotensin; a blood pressure regulating enzyme that has a role in controlling hypertension (Sang-Hoon Lee et al., 2010) Enzyme inhibitory activity against acetylcholinesterase and butylcholinesterase by phlorotannins were considered as a potent treatment for Alzheimer’s disease (Li et al., 2011) Phlorotannins as matrix metalloproteinase enzymes inhibitors were shown as potential components against metastasis, arthritis, chronic inflammation, wrinkle formation (Kim et al., 2006) The presence of free radicals can be one of the reasons for the formation of cancer cells in human body Phlorotannins act as free radical scavenging compounds that show potential to reduce cancer formation in human body (Li et al., 2011)

Polysaccharides, a class of macromolecules like alginates (alkali-soluble polysaccharides), fucoidans, laminarans (water-soluble polysaccharides) have been shown to possess various biological activities including antiviral, anti-inflammatory, anticoagulant, antiangiogenic and immunomodulatory activity (Wijesinghe & Jeon, 2012) Polysaccharides are not digested by intestinal enzymes, due to inter-chain hydrogen bonds and are considered to be

an important source of prebiotics, and dietary fibre that help reduce weight by prolonging the gastric emptying rate which lowers food intake and reduces the risk of obesity and colon

4

preventative actions of polysaccharides may include inhibitory effects against cancer cell proliferation, induction of tumor cell apoptosis, stimulation of immunity, inhibition of angiogenesis and inhibition of the tumor invasion through modulation of metalloproteinases Biological properties of polysaccharides may depend on the differences in the structures, molecular weight and algal species (Fedorov et al., 2013)

1.1.2 Bioactive compounds from brown algae

Numerous biologically active compounds have been identified in brown algae In this project however, three classes of compound; fucoxanthin, phenolics (phlorotannins) and polysaccharides (fucoidans) were selected for assessment of their antioxidant and cytotoxic properties for activity against pancreatic cancer cell lines

a) Fucoxanthin

Carotenoids, chlorophylls, and phycobiliproteins are three classes of pigment found in marine algae (Pangestuti & Kim, 2011) Carotenoids are classified into two subtypes: carotenes and xanthophylls (Batista et al., 2006) Fucoxanthin is a xanthophyll, with distinguishing chemical features including an unusual allene linkage and a three membered epoxide on the respective cyclohexane rings (Kumar et al., 2013) Almost all alkene

linkages in fucoxanthin isolated from brown algae possess a trans-configuration (Figure 1)

(Jaswir et al., 2013; Nakazawa et al., 2009) Fucoxanthins are one of the most abundant carotenoids found in nature, comprising approximately 10% of total carotenoid abundance (Rajauria et al., 2016)

Fucoxanthin is absorbed at the intestinal level with dietary fats and is metabolised mainly to fucoxanthinol in the gastrointestinal tract by digestive enzymes (lipase and cholesterol esterase) and subsequently to amarouciaxanthin A in the liver (Martin, 2015)

extract (Imbs et al., 2013)

b) Pholorotannins

Phlorotannins are phenolic polymers found in brown algae The base structural unit is a phloroglucinol ring (1,3,5-trihydroxybenzene) (Figure 2) Phlorotannins are classified into four subclasses based on subunit linkage characteristics; namely ether (fuhalols and phlorethols); phenyl (fucols); ether and phenyl linkage (fucophlorethols), and dibenzodioxin (eckols) (Singh & Bharate, 2006) Molecular weights of phlorotannins range from 126 kDa

to 650 kDa and are formed biosynthetically via the acetate-malonate pathway (Wijesekara & Kim, 2010) Phlorotannins derived from brown algae are considered to have a stronger free radical scavenger capacity than polyphenols derived from terrestrial plants They have up to eight interconnected rings in their structures, which compares with green tea catechins that have three to four interconnected rings (Mohamed et al., 2012)

Phlorotannins can comprise up to 15% of the dry weight of brown algae (Ragan & Glombitza, 1986; Targett & Arnold, 1998) There are a few chemical methods available for the analysis of phlorotannins reported in the literature This is due to phlorotannins being reactive and polar compounds and being large and structurally related to each other Colourimetric methods for estimation of total phenolic content (TPC) are most commonly used to quantify the proportion of phlorotannins present in algal extracts (Amsler & Fairhead, 2005)

Fucoidan is composed of sulfated L-fucose together with small proportions of xylose, mannose, D-galactose, L-rhamnose, arabinose, glucose, D-glucuronic acid (Pomin &

depending on the species, harvest season and extraction methods It was found that the low yields of polysaccharide fractions (0.30-0.96% algae dry weight) were observed from the

Sargassum pallidum extract (Liu et al., 2016), while with the alga Undaria pinnatifida

extract, it significantly varied from July (25.4–26.3%) to September (57.3–69.9%) (Mak et al., 2013) The molecular weight of the fucoidan also varies depending on the algal source

It is generally classified as low (< 10 kDa), medium (10 kDa-10.000 kDa) and high (> 10.000 kDa) (Matsubara et al., 2005) Variability in molecular weight, degree of sulfation, sulfate group position and monosaccharide composition in fucoidan polymers influences their biological activities (Senthilkumar et al., 2013; Yang et al., 2008b)

Information about structure and activity of sulfated polysaccharides from brown algae was well documented by previous studies Analysis of fucoidan content in the brown alga

Ascophyllum nodosum by high performance anion exchange chromatography showed the

main constituent proportions to be fucose (52.1%), galactose (6.1%), glucose (21.3%), and

and two main size fractions (47 kDa and 420 kDa) were observed by gel permeation

7

fucoidans possessed higher biological activity compared to native fucoidans Low molecular

anticancer activity with mild hydrolysis conditions using acid or microwave (Yang et al., 2008a) Lower molecular weight fucoidans and modification of the binding properties of

al., 2010) Sulfate content had a positive correlation with anti-proliferative efficacies of the polysaccharide fractions from tropical algae (Costa et al., 2010)

d) Other components

Beside the three main chemical components mentioned above, other constituents present in brown algae also possess various biological activities that exhibit positive health benefits, making them of interest for potential application in functional food and pharmaceutical industries

Bromophenols and other halogenated compounds from marine algae have been reported to possess a range of biological activities including antioxidant, antimicrobial, antiviral, anti-inflammatory, anticancer, anti-diabetic, and anti-thrombotic effects (Cabrita et al., 2010) Red algae possess higher concentrations of bromophenols in comparison to brown and green algae (Liu et al., 2011; Shi et al., 2009) Terpenoids and associated derivatives present in brown algae have been shown to possess anti-cancer activity (Sharma et al., 2016) Meroditerpenoids are comprised of a polyprenyl chain attached to a hydroquinone ring moiety and triterpenoids contained around thirty carbon atoms with acyclic squalene in their structures These compounds, which are common in brown algae, display antitumor activity (Li et al., 2013; Reddy & Urban, 2009) Marine algae are considered as a rich source of lipids having potential applications in many fields (Hossain et al., 2005)

8

halogenated indole alkaloids) Although marine organisms are rich in alkaloids, few have been found to possess anticancer activity (Güven et al., 2010)

1.1.3 Extraction of algal compounds

Extraction of natural products is a process whereby target compounds are isolated from plant or animal tissues The process is performed in a manner that ensures that the components of interest are not denatured or destroyed during extraction Extraction success

is based on chemical compatibility (like polarity) between the extracting solvent and target(s) Extraction from organic materials typically involves one or more of three techniques namely soxhlet extraction, maceration and hydrodistillation (Kadam et al., 2013) The yields and purity of the target compounds are two main factors for selecting appropriate extraction methods The equipment utilized in traditional extraction techniques is simple and inexpensive, but these methods have several major drawbacks including low extraction efficacy and high degradation of labile compounds due to prolonged exposure to high temperatures Extraction with organic solvents also typically requires large solvent to mass ratios, which raises environmental and health concerns regarding disposal (Azmir et al.,

advantages over conventional methods have been developed for extracting substances from marine algae and plants

a) Microwave Assisted Extraction

More novel techniques have also been applied to improve the extraction efficiency of active constituents from algae These include pressurised liquid extraction (PLE), supercritical fluid extraction (SFE), enzyme assisted extraction (EAE), ultrasound assisted extraction

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reduced levels of degradation of thermo-labile compounds (Michalak & Chojnacka, 2014)

Of these, MAE has been viewed as being particularly advantageous compared to traditional Soxhlet extraction (Polshettiwar & Varma, 2008), with rapid internal heating of the algal matrix based on solvent interaction with electromagnetic waves, resulting in rapid degradation of cellular structures leading to the liberation and solvation of target compounds (Zhang et al., 2011) Some disadvantages of MAE have also been noted including degradation of thermo-labile components as a consequence of high power microwave irradiation or required use of polar solvents (e.g water, methanol, acetonitrile) with high microwave absorption efficiency

Lin et al (2013) successfully employed MAE with aqueous ethanol as the solvent to

optimise the antioxidant activities of Monostroma nitidum extracts, while enhanced phlorotannin yield and anticancer activity was obtained from Saccharina japonica Aresch

extracts by MAE compared with traditional liquid extraction (He et al., 2013) The thermal stability of phenolic compounds indicated by Liazid et al (2007) that all phenolic standards and phenolics from grape skin and seeds were stable with temperature of up to 100 °C for

20 min under conditions of MAE, while there is significant degradation of epicatechin, resveratrol and myricetin at temperature of 125 °C The advantages and drawbacks of MAE extraction of plant materials has been reviewed in recent studies (Kala et al., 2016; Wang & Weller, 2006; Zhang et al., 2011)

b) Ultrasound Assisted Extraction

Ultrasound assisted extraction (UAE) is considered an effective extraction method in comparison with traditional technologies because of its low energy requirements and solvent consumption (Chemat & Khan, 2011) Ultrasound irradiation (20 kHz to 10 MHz) enhances extraction efficacy by propagating mechanical ultrasonic waves that improve solvent

UAE improved the extraction efficacy of high molecular weight phenolic compounds from

Ascophyllum nodosum (Kadam et al., 2015) and was used to isolate bioactive

polysaccharides from Sargassum fusiforme in higher yield The antioxidant activity of these

extracts was also found to be greater than conventional hot water extraction of the same material (Li et al., 2013) The advantages and drawbacks of UAE from plants have been highlighted in previous studies (Chemat & Khan, 2011; Romanik et al., 2007)

1.1.4 Isolation and identification of bioactive compounds

a) Chromatographic separation and isolation of bioactive compounds

Isolation of compounds in a pure state is a key aspect of assessing bioactivity of natural products The ability to isolate individual compounds or compound fractions depends on the physicochemical feature of target compounds including polarity, viscosity, thermal stability, solubility (hydrophobicity or hydrophilicity), acid-base properties, functional group profile and molecular weight (Nyiredy, 2004; Sticher, 2008) More than one isolation/purification method is often required to achieve appropriate purity, for example chromatographic purification may be employed in conjunction with physical methods such as distillation, crystallization or liquid-liquid isolation to achieve high state purity (Bucar et al., 2013) It is the relative balance in physical and chemical properties which generally determines the chosen purification pathway

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the separation of a mixture It consists a mobile phase (using a gas for gas chromatography,

a liquid for liquid chromatography, a liquid at critical temperature and pressure for

supercritical fluid chromatography), which carries the mixture through another material called a stationary phase The various constituents of the mixture travel at different speeds,

causing them to separate (Jandera, 2011; Taylor, 2009) In addition, based on the separation mechanism, chromatography is also divided into ion-exchange chromatography (uses an anionic or cationic stationary phase to separate ions and polar molecules based on their affinity to the ion exchanger and applied to charged molecule such as large protein, small nucleotides, and amino acids) (Jungbauer & Hahn, 2009), size-exclusion chromatography (molecules separated by their size and applied to macro-molecules) (Gellerstedt, 1992)

Normal-phase chromatography is performed using a hydrophilic stationary phase containing unmodified silica or alumina resins Therefore, molecules with hydrophilic properties in the mobile phase having a high affinity for the stationary phase are adsorbed to the column, while hydrophobic molecules, with less of an affinity for the column, will be eluted and detected first (Yoshida, 1997) In addition, the hydrophilic molecules adsorbed to the column are required to elute using polar solvents This column is suited to lipophilic compounds, and solvents used for this are n-hexane, chloroform, dichloromethane, diethyl ether, ethyl acetate (Yoshida, 2004) Alternatively, reversed-phase chromatography is a technique using a hydrophobic stationary phase, which has a stronger affinity for less polar compounds created by alkyl chains with covalent bonds (Dorsey & Dill, 1989) It is called reversed-phase chromatography due to the reverse of the polarity between the mobile and stationary phases As a result, hydrophilic molecules will pass the column first using a polar mobile phase, and then hydrophobic molecules with high affinity to the column will be

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chromatography can be applied to elute both hydrophilic and hydrophobic compounds

In my project, liquid chromatography was applied for further separation and purification of algal components Counter-current chromatography (partition) was used to create phenolic-enriched fractions Fucoxanthin and phenolics were identified by thin layer chromatography, while column chromatography was applied for isolation of fucoxanthin A HPLC system was used for identification and quantitation of fucoxanthin

• Counter-current chromatography

Counter-current chromatography (partition) is a liquid–liquid isolation technique in which both the mobile and the stationary phase are liquids The stationary phase is held in place by centrifugal force with the mobile phase flowing past Fractionation and isolation of individual compounds is based on partitioning between two immiscible solvents, with the proportion of the sample residing in each phase dependent on partition characteristics expressed and partition coefficients (Sticher, 2008)

The isolation of individual and groups of bioactive compounds from algae has been achieved using counter-current chromatography and found to be efficient, with differences

in yield and antioxidant activities of fractions enhanced when compared to crude extracts

For example, ethanol extracts from green algae Enteromorpha compressa, Capsosiphon

fulvescens, Chaetomorpha moniligera, and Ulva pertusa were redissolved in water and

partitioned sequentially with n-hexane (Hx), chloroform (CF) and ethyl acetate (EA) to generate four fractions, with the CF fraction found to contain the highest concentration of phenolics and possessing the highest antioxidant activity (Cho et al., 2010) In addition, five fractions - petroleum ether (PE), ethyl acetate (EA), dichloromethane (DCM), butanol

(BuOH) and water (AQ) were produced by partitioning methanolic extracts of Sargassum

marginatum, Padina tetrastomatica and Turbinaria conoides The highest concentration of

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fraction of S marginatum showed the highest DPPH scavenging activity (Chandini et al.,

2008) Using centrifugal partition chromatography (CPC) with a two-phase solvent system

of n-hexane–ethyl acetate–ethanol–water (5:5:7:3, v/v/v/v), fucoxanthin from Eisenia

bicyclis (Kjellman) Setchell (Laminariaceae) was separated in the system with the purity of

fucoxanthin obtained at 81% for the first CPC step and over 98% after the second step (Kim

et al., 2011)

• Thin layer chromatography

Thin layer chromatography is a separation technique that employs liquid mobile phase migration through a solid stationary phase either by capillary action (preparative thin-layer chromatography) or by rotational forced flow (rotation thin-layer chromatography), centrifugation (centrifugally accelerated thin-layer chromatography), or pressure (overpressure thin-layer chromatography) (Marston & Hostettmann, 1991) Numerous absorbent types may be employed for the solid phase matrix including silica gel, aluminium oxide, cellulose or starch The coating on the TLC plates and mobile phases are chosen depending on the properties of the target compounds (Sherma, 2000)

Fucoxanthin from the Himanthalia elongata non-polar solvent extract (equal-volume

mixture of n-hexane, diethyl ether and chloroform) has been isolated using thin layer chromatography with a mixture of chloroform/diethyl ether/n-hexane/acetic acid (10:3:1:1, v/v/v/v) as the mobile phase, with individual chromatographic bands containing compounds

of interest, including fucoxanthin (high purity ~97%), confirmed by HPLC analysis (Rajauria et al., 2016)

• Flash chromatography (FC)

Flash chromatography (FC) is a modified form of traditional gravity based column chromatography FC utilizes a glass column (100-200 mm) of moderate diameter (20 – 70

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stationary phase) is unusually loaded onto the column as a suspension Dry packing can be employed but requires the addition of a binding agent (such as gypsum) to improve packing characteristics and to preserve the structural integrity of the column in the dry state FC is employed for the separation of both lipophilic and hydrophilic substance mixtures (Roge et al., 2011) The individual components of a mixture are eluted from the column using a liquid mobile phase In normal phase chromatography, less polar compounds adhere less strongly to the polar silica and elute fastest The polarity of the mobile phase is therefore typically graduated (non-polar to polar) during elution Gravity based elution is used in traditional column chromatography but this can be accelerated using positive gas pressure (nitrogen or compressed air) applied to the top of the column In the case of dry packed FC columns, negative pressure can be utilised to “pull” the solvent through the stationary phase (Bucar et al., 2013) In all cases, solvent fractions are collected at regular intervals and assessed by TLC for purity, with common fractions then combined to yield a pure product

FC is a simple, cheap and common separation technology for isolating individual compounds from complex mixtures

A dichloromethane fraction from the Sargassum siliquastrum methanol extract was applied

to a column loaded with silica gel 60 (70-230 mesh, Merck) The mixtures of chloroform and methanol with the ratios: 99:1, 95:5, 90:10, 80:20, and 50:50 have been used as a mobile phase to elute four sub-fractions successively that possessed phenolic compounds with different antioxidant and lipid peroxidation inhibitory activities (Lim et al., 2002)

Fucoxanthin was successfully separated from the Sargassum binderi and S duplicatum

benzene fraction through a chromatography column with n-hexane/acetone (6/4; V/V) and acetone as mobile phases with the purity of fucoxanthin of over 90% achieved (as validated

by HPLC analysis) (Jaswir et al., 2012)

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High-performance liquid chromatography (HPLC) has become a significant tool for the isolation, purification, quantification and identification of most classes of natural products, and is usually the final step of the purification process (Sasidharan et al., 2011) A HPLC instrument typically includes a degasser, a sampler, pumps, and a detector Smaller sample volumes injected, and the smaller size of the column filled with smaller sizes of absorbent particles makes it highly able to distinguish between compounds This leads HPLC to be a popular chromatographic technique compared with traditional chromatography (Tsao & Deng, 2004)

For example, in separation of phlorotannins from Sargassum ringgoldianum, a bifuhalol (a

phenolic compound) from the phlorotannin fraction was isolated by RP-HPLC and identified by nuclear magnetic resonance (NMR) and mass spectra (MS) (Nakai et al.,

2006) Fucoxanthin content from the brown alga Undaria pinnatifida (Fung et al., 2013) and

a marine micro-alga, Chaetoceros calcitrans (Foo et al., 2017) was identified and quantified

by HPLC analysis A HPLC system was also applied to investigate properties of three

polysaccharide fractions from Opuntia milpa alta (Cai et al., 2008)

b) Identification of bioactive compounds

High-performance liquid chromatography (HPLC) is also a tool for detection of natural components In recent years, a combination of a separation technique with one or more spectroscopic detection techniques, known as hyphenated technique, has been employed to great success This technique has been demonstrated to be effective for both qualitative and quantitative analysis of unknown compounds in complex natural extracts or fractions (Sforza et al., 2006) Structural information of the compounds present in a crude sample is identified by a analysis system via a link of a high-performance liquid chromatography (HPLC), gas chromatography (GC) with spectroscopic detection techniques (Fourier-

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spectroscopy (MS), and nuclear magnetic resonance spectroscopy (NMR), resulting in various modern hyphenated techniques (LC-MS, GC-MS, LC-PDA, and LC-NMR or more than two techniques such as LC-PDA-MS, LC-MS-MS, LC-NMR-MS, LC-PDA-NMR-MS) (Cai et al., 2002; Rauter et al., 2005; Ye et al., 2007) The connection of HPLC and MS

or NMR has increased the capability of solving structural problems of complex natural products

1.1.5 Activities of algal compounds against several cancer cell lines

Fucoxanthin, phlorotannins, sulfated polysaccharides (fucoidans) and other components have been demonstrated to be effective against several cancer cell lines The anticancer activity of a number of algal compounds has been postulated through several different mechanisms, including anti-proliferation, induction of apoptosis, cell cycle arrest and anti-angiogenesis, immunomodulatory effects, antimitogenic activity, and anti-cell migration effects

Fucoxanthin exhibits anti-proliferative potential against several malignancies, including prostate cancer (Kotake-Nara et al., 2005), human leukemia HL-60 (Kim et al., 2010), colon cancer (Hosokawa et al., 2004), urinary bladder cancer (Zhang et al., 2008), gastric cancer (Yu et al., 2011), breast cancer (Konishi et al., 2006) and melanoma (Kim et al., 2013) in a dose dependent manner The anti-proliferative activities of fucoxanthin could be mediated through the up-regulation of the p21WAF1/Cip1, ROS-mediated Bcl-xl pathway, down-regulation of the cyclin D, JAK/STAT signal pathway, and is linked with GADD45a, p38 MAPK and SAPK/JNK (Rengarajan et al., 2013) In HL-60 cells, fucoxanthin causes cleavage of procaspase-3 and poly-ADP-ribose polymerase, and mediates apoptosis induction through mitochondrial membrane permeabilization and caspase-9 and caspase-3