Brown algae as a source of bioactive

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

i 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

ii 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.

iii 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, Bowyer MC, Van Altena IA & Scarlett CJ. (2018). Comparison of chemical profile and antioxidant properties of the brown algae, International Journal of Food

Science & Technology 51(1): 174-181. doi: 10.1111/ijfs.13571.

2. Paper II: Dang TT, Vuong QV, Schreider MJ, Bowyer MC, Van Altena IA & Scarlett CJ.

(2017). The Effects of Drying on Physico-Chemical Properties and Antioxidant Capacity of the

Brown Alga (Hormosira banksii (Turner) Decaisne). Journal of Food Process and

Preservation 41(4): e13025. doi.org/10.1111/jfpp.13025.

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.

Journal of Applied Phycology 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 of the brown alga Sargassum vestitum. Separation Science and Technology (In Press). 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 BotanicaMarina.

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

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 Biomedicineand Pharmacotherapy.

v 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 “Comparison of chemical profile and antioxidant properties of the brown

algae”, International Journal of Food Science & Technology 51(1): 174-181.

doi:10.1111/ijfs.13571.

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:

vi 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)”. Journal of Food Process and Preservation 41(4): e13025. doi.org/10.1111/jfpp.13025.

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.

vii 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”. Journal of Applied

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

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: viii 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

of the brown alga Sargassum vestitum” Separation Science and Technology (In Press).

doi.org/10.1080/01496395.2017.1414845.

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:

ix STATEMENT OF AUTHORS' CONTRIBUTION TO THE PAPERS

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.

x STATEMENT OF AUTHORS' CONTRIBUTION TO THE PAPERS

To whom it may concern,

We, Deep J. Bhuyan, Danielle R. Bond, 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 “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.

xi STATEMENT OF AUTHORS' CONTRIBUTION TO THE PAPERS

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.

xii STATEMENT OF AUTHORS' CONTRIBUTION TO THE PAPERS

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:

xiii 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. Oralpresentation.

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

antioxidant capacity of the brown alga (Hormosira banksii). 3rd International Conference

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

Vietnam. Oralpresentation.

xiv LIST OF ABBREVIATIONS

°C Degree Celsius μL Microliter (s) ANOVA Analysis of Variance AQ Aqueous fraction BuOH Butanol fraction DCM Dichloromethane fraction EA Ethyl acetate fraction et al. and others g gram (s) Hx Hexane fraction h hour( s) MAE Microwave Assisted Extraction min Minute (s) mL Millilitre (s) Mw Molecular weight nM Nano-moles RP-HPLC Reversed-phase High Performance Liquid Chromatography RSM Response Surface Methodology sec Second (s) TFC Total Flavonoid Content TLC Thin Layer Chromatography TPC Total Phenolic Content UAE Ultrasonic Assisted Extraction UV-Vis Ultraviolet- Visible v/v Volume by Volume w/v Weight by Volume w/w Weight by Weight W Watt (s)

xv FIGURES FOR THESIS

Figure 1: Trans-fucoxanthin

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

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

Structure of fucoidan unit

Figure 3: Sulfated polysaccharides (Fucoidans)

xvii TABLE OF CONTENTS

STATEMENT OF ORIGINALITY …………………………………………………………i DECLATATION OF AUTHORSHIP………………………………………………………ii ACKNOWLEDGEMENTS………………………………………………………………...iii LIST OF PUBLICATIONS INCLUDED AS PARTOF THE THESIS…………………....iv STATEMENTS OF AUTHOR S’ CONTRIBUTIONTOTHE 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...... 5 b) Pholorotannins...... 6 c) Polysaccharides (fucoidans)...... 7 d) Other components ...... 9 1.1.3 Extraction of algal compounds...... 10 a) Microwave Assisted Extraction ...... 10 b) Ultrasound Assisted Extraction...... 11 c) Overview of Response Surface Methodology (RSM)………………………….12 1.1.4 Isolation and identification of bioactive compounds ...... 13 a) Chromatographic separation and isolation of bioactive compounds ...... 13 b) Identification of bioactive compounds...... 18 1.1.5 Antioxidant activity and activities of compounds against several cancers ...... 19 a) Antioxidant activity of algal compounds in brown algae…………………...... 19 b) Activities of compounds against several cancer cell lines……………………...20 1.1.6 Algal components against pancreatic cancer cell lines ...... 22 a) Problems with pancreatic cancer...... 22 b) Algal compounds against pancreatic cancer ...... 23 1.2 Research content ...... 25 1.3 Research Aims and Expected Outcomes ...... 26 1.4 Experimental Rationale...... 27

xviii

1.5 Hypothesis, aims and objectives...... 28 PART 2: RESULTS ...... 31 2.1 Synopsis of research papers published from results ...... 31 2.2 Research papers published from results...... 37 PART 3: GENERAL DISCUSSION AND CONCLUSIONS...... 39 3.1 General discussion ...... 39 3.1.1 Preparation of the samples (drying and extraction) ...... 40 3.1.2 Bioactive compounds and isolation processes from brown algae...... 44 3.1.3 Activity of algal compounds against pancreatic cancer cell lines...... 47 3.2 Conclusions and recommendations...... 50 3.2.1 Conclusions...... 50 3.2.2 Recommendations...... 52 BIBLIOGRAPHY ...... 53

xix

ABSTRACT

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.

xx The overall aims were to:

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

power.

x Optimise the extraction of phenolics from algae using irradiative technologies such

as ultrasound and microwave.

x Isolate key components including fucoxanthin, phenolics and polysaccharides for

assessment of their cytotoxic activity against a range of pancreatic cancer cell lines.

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 temperature of 50 °C was found to be optimal for both de-humiditification and vacuum oven 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) with 158.82 mg GAE.g-1 followed by S. vestitum and Padina sp. (141.91 and 124.65 mg

GAE.g-1, respectively). Total flavonoid content (TFC) was highest in H. banksii (29.31

CAE.g-1), while Padina sp. produced the highest tannin content (56.17 mg CAE.g-1).

Fucoxanthin 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 determined to be; temperature = 30 °C, extraction time = 60 min., irradiation power =

60% (150w). The highest values of TPC and antioxidant activity (ABTS, DPPH and FRAP)

xxi 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.

Microwave-assisted extraction was applied to extract phenolics from S. vestitum using aqueous 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-1 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 of concentrations (25 – 200 ng.mL-1), phenolic

xxii compounds possessing medium polarity (ethyl 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 (HPDE). It was noted that polar phenolic compounds (butanol fraction) exhibited high cytotoxic activity against these cancer cells but low toxicity against non-tumorigenic cells. Fucoxanthin was also a potent agent against pancreatic cancer cell lines with high growth inhibition (inhibition of 30.91–

92.81% at concentrations of 100–200 μg.mL-1). Polysaccharides fractions (CF50 and CF70) showed quite high activity (inhibition of 39.35 – 82.82% at the concentrations of 100–200

-1 μg.mL ) 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.

xxiii PART 1: LITERATURE REVIEW

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 GLQRÀDJHOODWHV 'LQRSK\FHDH  (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 1 benefits in the healthcare industry (Kadam & Prabhasankar, 2010). The chemical and 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).

Hormosira banksii is often dominant in the lower eulittoral and sublittoral zone of rocky shores and the muddy tidal flats of estuaries in southern Australia (Kevekordes & Clayton,

2000; Myers et al., 2007; Ralph et al., 1998; Underwood, 1998; Vuong et al., 2018).

Sargassum vestitum (R. Brown ex Turner) C. Agardh (Phaeophyta), a common intertidal dioecious brown alga, is widely distributed in southern Australia (May & Clayton, 1991;

Steinberg, 1994), while Sargassum linearifolium (Turner) C. Agardh was collected at Shark

Bay, Port Jackson, New South Wales, Australia reported by Poore et al. (2000). Padina sp. was collected in Pioneer Bay on the leeward side of Orpheus Island (18° 35’ S, 146° 20’ E) on the inner shelf of the Great Barrier Reef, Australia (Mantyka & Bellwood, 2007).

Phyllospora comosa, a monotypic, perennial fucoid alga, is commonly found on the shallow subtidal reefs of temperate south-eastern, and Tasmania of Australia (Coleman et al., 2008;

Marzinelli et al., 2014; Valentine & Johnson, 2004). Sargassum podocanthum was also reported in diversity of benthic macroalgal assemblages in south-western, Australia

(Kendrick et al., 2004). All these species in my thesis were collected in March, 2016 from the rocky shore at Bateau Bay, NSW, Australia (Latitude of 33°22'55.2"S; longitude of

151°29'6"E).

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 2 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

KK-Ay mice (Maeda et al., 2007). Fucoxanthin also promotes energy released as heat from 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 treating free radical-related diseases (Ngo et al., 2011). Biological activity of phlorotannins 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.

Phlorotannins display inhibitory effects against a range of important enzymes including Į- glucosidase as anti-diabetic activity (Iwai, 2008)Į-JOXFRVLGDVHDQGĮ–amylase (responsible for the digestion of oligosaccharides, glucose absorption and the maintenance of glucose levels in plasma, leading to the suppression of postprandial hyperglycemia) (Thomas & 3

Kim, 2011). 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 (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 cancer (Gupta & Abu-Ghannam, 2011). Molecular mechanisms behind the cancer 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

Brown algae bioactives include polysaccharides (insoluble and soluble dietary fibres (Holdt

& Kraan, 2011)); phenolics (phlorotannins); pigments (chlorophylls, carotenoids, phycobiliproteins, and xanthophylls) (Aryee et al., 2018) and proteins and amino acids.

Essential amino acids are observed in most algal species and represent a rich source of these 4 compounds, particularly aspartic acid and glutamic acid. Algal proteins are found in higher concentration than in terrestrial plants (Balbao et al., 2013; Holdt & Kraan, 2011). Lipid content in marine brown algae is low (0.57–3.5% dry weight), and algal lipids have a higher proportion of polyunsaturated fatty acids (PUFA) compared with terrestrial plants. Terpenes are lipophilic secondary metabolites can be grouped into hemi-, mono-, sesqui-, di-, sester-, triterpenes together with tetraterpenoids (carotenoids) (Balbao et al., 2013).

In this thesis 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) &DURWHQRLGV DUH FODVVL¿HG LQWR WZR 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).

Fucoxanthin extraction from algae is achieved in organic solvents of mid to low polarity, with concentration enriched by solvent partition techniques and purification by column chromatography, centrifugal partition chromatography or HPLC (Kim et al., 2011). 5

Fucoxanthin content in Padina australis has been reported to be 0.04%, lower than two other brown algae Laminaria digita (2.4%) and Padina tetrastromatica (1.7%) (Jaswir et al., 2013). Ethanol extraction of Fucus evanescens, fucoxanthin yielded 0.05% of the crude extract (Imbs et al., 2013).

b) Pholorotannins

Phlorotannins are phenolic polymers found in brown algae and constitute an extremely heterogeneous group of molecules which provides wide ranging biological activity (Holdt &

Kraan, 2011). The base structural unit of phlorotannins is the phloroglucinol ring (1,3,5- trihydroxybenzene) (Figure 2). Phloroglucinols are comprised principally of monomeric units and incorporate a range of derivatives including acyl phloroglucinols, phloroglucinol– terpene adducts, phloroglucinol glycosides, halogenated phloroglucinols, prenylated/geranylated phloroglucinols, phloroglucinols linked to a D-pyrone ring and cyclic polyketides. Dimeric systems comprised of two phloroglucinol units joined either through a methylene linakge or by the formation of a chroman ring, trimerics such as filicinic acid, aspidinol, phloroaspin, desaspidin, albaspidin, fixilic acid, tetramerics and phlorotannins also exist.

Phlorotannins are FODVVL¿HG LQWR IRXU VXEFODVVHV EDVHG RQ 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). They possess a variety of metabolic roles, including both primary (cell wall construction, storage and in reproduction) and secondary (herbivore defense and UV protection) (Fairhead et al., 2005). 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 6 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).

c) Polysaccharides (fucoidans)

Polysaccharides are polymers produced from monosaccharides units linked via glycosidic bonds. They serve a range of purposes in natural systems from structural support to energy storage and are commercially important materials in food manufacturing forming the basis of stabilizers, thickeners and emulsifiers in food, feed and beverage production (Wu et al.,

2016). Marine algae are a rich source of both sulfated and non-sulfated polysaccharides.

Non-sulfated polysaccharides include Laminarin, which is used in energy storage, while alginic acid (alginate) is an important structural carbohydrate. The main sulfated polysaccharides found in brown seaweeds are the fucose based fucans such as fucoidan and ascophyllan. Fucoidan (Figure 3) is a sulfated-polysaccharide which has been most frequently investigated for its antioxidant and anticancer activity (Xue et al., 2012; Yang et al., 2013).

Fucoidan is composed of sulfated L-fucose combined with small proportions of D-xylose,

D-mannose, D-galactose, L-rhamnose, arabinose, glucose, D-glucuronic acid (Pomin &

Mourão, 2008; Wu et al., 2016). Yields of fucoidans from brown algae have widely varied,

7 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 xylose (16.5%), while sulfate content was determined to be 19% by CaCl2 gelatin method and two main size fractions (47 kDa and 420 kDa) were observed by gel permeation chromatography analysis (Foley et al., 2011). In addition, it was shown that low weight fucoidans possessed higher biological activity compared to native fucoidans. Low molecular weight fucoidan by hydrolysis (Mw = 490 kDa) from 8QGDULD SLQQDWL¿GD enhanced 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 with sulfate groups from 8QGDULDSLQQDWL¿GD improved their anticancer activities (You et al., 2010). It has been shown that different sulfate patterning (both sulfate content and position within the sugar polymer chain) of marine polysaccharides affects antitumor activity (Kasai et al., 2015). Sulfate content has a positive correlation with the anti- proliferative efficacy of polysaccharide fractions isolated from tropical algae (Costa et al., 8

2010). Highly sulfated fucoidan has higher antiangiogenic activity than native fucoidan

(Koyanagi et al., 2003). Ale et al. (2011). The anti-cancer activity of fucoidan was found to be linked to sulfate content rather than monosaccharide content by comparing the effect of fucoidan from two algal species on Lewis lung carcinoma and melanoma B16 cells.

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).

Alkaloids in marine algae occur as three groups; phenylethylamines, indoles and halogenated indole alkaloids). Although marine organisms are rich in alkaloids, few have been found to possess anticancer activity (Güven et al., 2010).

9

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

HI¿FDF\ 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.,

2013; Polshettiwar & Varma, 2008)7KHUHIRUHQHZH[WUDFWLRQWHFKQLTXHVZLWKVLJQL¿FDQW 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

ÀXLG H[WUDFWLRQ 6)(  HQ]\PH DVVLVWHG H[WUDFWLRQ ($(  XOWUDVRXQG DVVLVWHG H[WUDFWLRQ

(UAE), and microwave assisted extraction (MAE) (Joana Gil-Chávez et al., 2013).

Advantages of these methods include higher yields, shorter extraction times, lower cost and 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 10 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 penetration into plant materials and increasing the contact surface area between solid and liquid phase. Moreover, cavitation phenomena (microbubble formation and implosion) generated by ultrasonic irradiation, leads to rapid degradation of cellular structures resulting in enhanced mass-transfer to the solvent phase, accelerated kinetics and reduced damage to 11 thermo-labile compounds (Macías-Sánchez et al., 2009; Teh & Birch, 2014; Wang et al.,

2008). UAE has wider applicability than MAE because it does not impose restrictions on solvent character (Kadam et al., 2013).

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).

c) Overview of Response Surface Methodology (RSM)

Response Surface Methodology (RSM) is the most popular statistical tool for the optimisation of the chemical and biochemical processes. RSM is a collection of statistical and mathematical techniques based on the fit of empirical models to experimental data obtained through experimental design %Dú %R\DFÕ . RSM can optimise the yield of experimental processes by reducing the number of iterative experimental trials required.

Preliminary experiments are required to determine independent variables in equations controling outputs in the experimental process. Determination of the levels of independent variables significantly affects the success of optimisation process (Bezerra et al., 2008).

Key stages in the application of RSM as an optimization technique are: (i) the selection of independent variables of variables and their levels; (ii) the experimental design is chosen and the experiments are conducted according to the experimental matrix. The mathematic– statistical models are estimated; and the predicted experimental data are evaluated and verified for the model’s fitness. From that, the optimum values for each studied variable are obtained.

RSM generates significant data from a small relatively small number of iterative 12 experiments and predicts interactions between independent parameters based on the the results of classical experiments. However, it is required to make preliminary work to choose independent variables and their levels. In addition, it is hard to say that all systems containing curvature are well accommodated by the second order polynomial %Dú 

%R\DFÕ .

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.

Principles of Chromatographic Separation: Chromatography is a technique for 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. 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) 13

(Jungbauer & Hahn, 2009), size-exclusion chromatography (molecules separated by their size and applied to macro-molecules) (Gellerstedt, 1992).

In my thesis, 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.

x Counter-current chromatography

Counter-current chromatography (partition) is a liquid–liquid isolation technique in which both the mobile and the stationary phase are liquids. Separation of individual compounds is based on partitioning between two immiscible solvents. In this method, one of the liquid phases operates as the stationary phase while other phase is pumped through the column as the mobile phase. Separation is achieved based on the differing partition coefficients of the solute within the two phases (Berthod et al., 2009). The partition coefficient is defined as the ratio of solute distributed between the mutually equilibrated two solvent phases (Ito, 2005).

Counter-current chromatography can reduce sample loss through irreversible solute absorption. This method can be directly applied to a crude extract with high efficacy, high recovery and low solvent consumption and solute loading capability. It is also suitable for separation of high polarity or unstable compounds, which is considered advantageous compared with conventional chromatography techniques (Huang et al., 2016).

The isolation of individual and groups of bioactive compounds from algae have 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 14 partitioned sequentially with 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 phenolics in this instance was present in the aqueous fraction of T. conoides, while the EA 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).

x 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).

From the Undaria pinatifida, fucoxanthin in the ethyl acetate fraction was isolated via preparative silica gel thin layer chromatography with a staged mobile phase

(chloroform:methanol:water; 65:25:4 v/v/v) followed by hexane:acetone; 6:4 v/v for further 15 purification (Hosokawa et al., 1999). 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).

x Flash chromatography (FC)

Flash Chromatography (FC) is a modification of conventional column chromatography.

Flash chromatography is an alternative to slow and often inefficient gravity-fed chromatography. The principle of FC is that the eluent is pushed through a short glass column under gas pressure (usually nitrogen or compressed air) (Sticher, 2008). The pressure of FC is lower than that of medium pressure liquid chromatography systems with a similar loading capacity. FC is a preferred separation technique due to its simplicity and low cost (Hobbs & Young, 2013).

FC utilises a glass column (100-200 mm) of moderate diameter (20 – 70 mm) fitted with a glass plug or sintered glass filter. Silica gel (typically used as the 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 16 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.

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).

x High-performance liquid chromatography (HPLC)

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. Nowadays,

There is increasing need to know natural bioactive components from the plants.

Conventional chromatographic techniques such as paper chromatography, thin layer chromatography and column chromatography lack the diagnostic capability of more sophisticated instrumental techniques such gas chromatography. This leads HPLC to be a popular chromatographic technique compared with traditional chromatography (Tsao &

Deng, 2004).

17

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- transform infrared (FT-IR), photodiode array (PDA) UV–Vis absorbance, mass 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.

18

1.1.5 Antioxidant activity and activities of compounds against several cancers

a) Antioxidant activity of algal compounds in brown algae

Oxidation is a chemical reaction that can produce free radicals, which can damage celluar structures. Antioxidant activity is an action that slows or prevents oxidative processes.

Antioxidant compounds can be classified into two sub-groups: “primary antioxidants”

(inhibiting oxidation reactions) or “secondary antioxidants” (inhibiting oxidation indirectly, by mechanisms such as oxygen-scavenging) (Craft et al., 2012). Antioxidants act in a number of ways including; (i) scavenging species responsible for initiation, (ii) interrupting the chain reactions, (iii) singlet oxygen quenching, (iv) synergism increasing the activity of chain-breaking antioxidants, (v) reducing action, (vi) metal chelation or (vii) inhibiting specific oxidative enzymes. Antioxidants often act by mixed and cooperative mechanisms

(Balbao et al., 2013).

Antioxidant activity of algal components was estimated using three antioxidant assay techniques; (i) ABTS - based on the scavenging ability of antioxidants to the radical anion

ABTS*+ (Thaipong et al. 2006); (ii) DPPH assay – which estimates antioxidant capacity via the ability of a compound to scavenge DPPH radicals (Brand-Williams et al. 1995); and ferric reducing antioxidant power (FRAP assay) based on the ability of an antioxidant compound to reduce a ferric oxidant (Fe3+) to a ferrous complex (Fe2+) by electron-transfer, and this indicated the capacity of the compound to reduce reactive species (Benzie & Strain,

1999).

Phenolic compounds can act as antioxidants by chelating metal ions, preventing radical formation and improving the antioxidant endogenous system (Cox et al., 2010). A higher ratio of phlorotannins was observed in brown algae compared to red and green algae, and these phenolics are the dominant antioxidants in brown algae (Balboa et al., 2013). The

19 radical scavenging capacity of polysaccharides in brown algae has been highlighted in previous studies (Camara et al., 2011; Hou et al., 2012; Zhang et al., 2010). It is proposed that scavenging ability is dependent on sulfate content, with highly sulfated polysaccharides exhibiting higher scavenging activity than low sulfated polysaccharides (Costa et al., 2010).

Fucoxanthin is the dominant carotenoid in brown algae, that has attracted significant recent attention due to its strong antioxidant properties and significant anti-cancer, anti-obesity and anti-inflammatory effects (Fung et al., 2013). It is suggested that fucoxanthin acts as a radical scavenger via transfer of the excited electron to its conjugated bonds to yield a more stable free radical and an excited state of carotenoids (Foo et al., 2015). Fucoxanthin is also considered as main antioxidant in the Hijikia fusiformis extract (Yan et al., 1999).

b) Activities of 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

20

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 activation (Kotake-Nara et al., 2005). Further, accumulation of reactive oxygen species

(ROS), deactivation of the Bcl-xL signalling pathway, induction of caspase-3, -7, and poly-

ADP-ribose polymerase cleavage that activates apoptosis of HL-60 cells (Kim et al., 2010).

Yu et al. (2011) reported that fucoxanthin down-regulated the expressions of CyclinB1 and survivin, inducing cell cycle arrest in G2/M phase and apoptosis in gastric adenocarcinoma

MGC-803 cells, while the reduction of Cyclin B1 by fucoxanthin through the JAK/STAT signalling pathway inhibited proliferation of MGC-803 cells. The anti-angiogenic effects by carotenoids on human umbilical vein endothelial cells was observed by Ganesan et al.

(2013), with fucoxanthin down-regulating signal transduction pathways by fibroblast growth factor 2 (FGF-2) and its receptor (FGFR-1).

Phlorotannin, isolated from brown alga Laminaria japonica, has shown considerable antiproliferative activity in the hepatocellular carcinoma cell line (BEL-7402) and on the murine leukemic cell line (P388) in a dose dependant manner (Yang et al., 2010). The induction of apoptosis was observed by a significant increase in the accumulation of the

Sargassum muticum polyphenol-rich extract-treated cells at sub-G1 phase via cell cycle analysis.

Fucoidan from the Cladosiphon okamuranus extract induces apoptosis of breast cancer cells

(MCF-7) via the caspase-8-dependent pathway with chromatin condensation, inter- nucleosomal DNA fragmentation, cleavage of poly (ADP ribose) polymerase, and activation of caspase-7, caspase-8, and caspase-9 (Yamasaki-Miyamoto et al., 2009). The induction of apoptosis of human colon cancer cells (HT-29 and HCT116) mediated through both the mitochondria-mediated and death receptor-mediated pathways was found from HT-29 and 21

HCT116 treated with fucoidan (0–20 ߤg/mL) from the Fucus vesiculosus extract (Kim et al.,

2010). Fucoidan was demonstrated to enhance the immune response by inhibitory effects against the growth of Ehrlich ascites carcinoma in mice with no sign of toxicity and increasing the phagocytosis activity of macrophages (Itoh et al., 1993). In another in vivo study, fucoidan from Fucus evanescens indicated anti-metastatic and antitumour activity in mice transplanted with Lewis lung adenocarcinoma cells (Alekseyenko et al., 2007). The high number of sulfate groups in the fucoidan molecule enhances the potency of its reported anti-angiogenic and antitumour activities (Koyanagi et al., 2003).

1.1.6 Algal components against pancreatic cancer cell lines

a) Problems with pancreatic cancer

Pancreatic cancer is the fourth leading cause of cancer related death with a 5-year survival rate of less than 7% (Bayraktar et al., 2010). The symptoms of this disease are not clear at the early stage, leading to late diagnosis. Besides, aggressive local invasion, early metastases, the limitations of surgical resection, and strong resistance to chemotherapy and radiotherapy are big challenges for the treatment of pancreatic cancer (Li et al., 2010).

Surgery is the only potential protocol for early disease stage, however resection rate is low, with only 15%-20% of patients are eligible for surgical resection (Verbeke et al., 2015).

Therefore, for patients with unresectable pancreatic cancer, chemotherapy and radiotherapy play a vital role in the treatments.

Gemcitabine (Gem) and 5-ÀXRURXUDFLO -FU)-based chemo-UDGLRWKHUDSLHVZHUHLGHQWL¿HG as the two main treatment options for locally advanced stage PC. However, in advanced metastatic stage PC, Gem has replaced 5-FU as the standard treatment for metastatic pancreatic cancer due to the longer overall survival time and apparent clinical improvements in the three common symptoms of pain, functional impairment, and weight loss (Burris et

22 al., 1997). To date, Gemcitabine is still considered as the standard treatment for pancreatic cancer patients. However, the Gem based chemo-resistance in pancreatic cancer is still hard to overcome in treatments (Hung et al., 2012). The combination of gemcitabine with numerous agents (5-fluorouracil, cisplatin, oxaliplatin, epirubicin, irinotecan, capecitabine, docetaxel, and mitomycin C), have been estimated in order to improve the therapeutic efficacies, but no clear benefits for overall survival were observed, while these agents were found to be toxic to the non-tumorigenic cells (Xu et al., 2011). From the problems above, it is clearly necessary for the development of novel and effective therapeutic strategies for the treatment of pancreatic cancer.

b) Algal compounds against pancreatic cancer

Studies have demonstrated the potential application of algal components for use against several pancreatic cancer cell lines. It was reported by McCauley et al. (2015) that in four species: Hormosira banksii (the EA extract), Phyllospora comosa (the DCM extract),

Solieria robusta (the EA and DCM extracts) and Ulvan sp. (the EA and DCM extract) showed high cytotoxic activities (76-100%) against pancreatic cancer (MiaPaCa-2) cells.

The Ulva sp. and H. banksii ethyl acetate extracts had selective cytotoxicity towards the pancreatic cancer cell line with no toxicity towards a normal murine cell line at 100 μg.mL-

1. The cytotoxicity of the Fucus vesiculosus acetone extract (Fv1) was evaluated on pancreatic cancer cell lines (panc1; panc98; pancTU1), revealing that Fv1 strongly inhibited the growth of the different tumour cell lines with the EC50 values (effective half maximal

FRQFHQWUDWLRQ  RI )Y  ȝJP/-1 IRU 3DQF78  ȝJP/-1 for Panc89; 1 and 9.23

ȝJP/-1 for Panc1. Importantly, Fv1 showed low cytotoxic activity against non-malignant resting T cells (Geisen et al., 2015).

23

The phenolics from several brown algae were investigated for their biological activities against pancreatic cancer cells. Several pancreatic cancer cell lines (MiaPaCa-2, Panc 3.27,

Panc 1 and BxPC-3) were used to assess the activities of the organic fraction of brown algae. The results indicated that almost all polyphenol enriched fractions (the DCM and EA fractions) of these algae showed high anti-proliferative activities (50–75%) against pancreatic cancer cells in vitro. In addition, apoptotic characteristics were observed via differential DNA fragmentation in cancer cell lines treated with polyphenol enriched fractions (Aravindan et al., 2013). The anti-proliferative activity of fucoidan from a brown alga Turbinaria conoides against MiaPaCa-2 and Panc-1 was evaluated with fucoidan significantly inhibiting both cancer cell lines in a dose-dependent and time-dependent manner. Apoptosis was observed via the appearance of apoptotic bodies characterised by chromatin condensation and nuclear fragmentation. The bioactive tetrapyrrole component, such as chlorophyll from Spirulina platensis showed anti-proliferative activities against pancreatic cancer cell lines (PA-TU-8902, MiaPaCa-2 and BxPC-3) and xenotransplanted nude mice .RQtFþková et al., 2014).

There are natural products from other marine sources that show potent activity against pancreatic cancer. The batzellines, are pyrroloiminoquinone alkaloids obtained from the

Caribbean sponge Batzella sp (family Esperiopsidae, order Poecilosclerida) that exhibit selective cytotoxicity towards the pancreatic cancer cell lines AsPC-1, Panc-1, BxPC-3, and

MIA PaCa2 (Guzmán et al., 2009). Manzamine A (a member of the manzamine alkaloids, was originally isolated from marine sponges of the genus Haliclona) can inhibit autophagy that is essential for pancreatic tumor growth by preventing autophagosome turnover, showing potential for the treatment of pancreatic cancer (Kallifatidis et al., 2013). Activity of butenolides, DUH D IDPLO\ RI Įȕ-unsaturated lactones, from a marine-derived fungus

Aspergillus terreus with antitumor activities against pancreatic ductal adenocarcinoma cells 24

(PANC-1 cell line) were observed via apoptotic body formation, membrane blebbing, cell shrinkage and nuclear condensation of cancer cells (Pimentel-Elardo et al., 2010; Qi et al.,

2018). Leiodermatolide, a polyketide macrolide isolated from a sponge of the genus

Leiodermatium, exhibits potent and selective cytotoxicity toward the pancreatic cancer cell lines AsPC-1, PANC-1, BxPC-3, and MIA PaCa-2, and potent cytotoxicity against skin, breast and colon cancer cell lines (Guzmán et al., 2016). Lasonolide A, a novel polyketide- derived macrolide, was previously isolated from an extract of the marine sponge Forcepia sp. and exhibited potent against pancreatic cancer (Isbrucker et al., 2009). Sansalvamide A, a natural product isolated from a marine fungus (Fusarium ssp.), exhibits antitumor activity on several cancer cell lines (Pan et al., 2009). While many agents from marine organisms have entered clinical trials, only four have been approved for use in humans, including

Cytarabine [AraC (cytosine arabinoside)], Yondelis® (ET743; trabectedin), Eribulin

(Halaven®, a synthetic derivative based on the structure of halichondrin B), and the ADC

Adcetris® (brentuximab vedotin) (Cragg & Pezzuto, 2016). There are no reports about the application of four compounds above for pancreatic cancer.

1.2 Research content

The research content is divided into the following three sections:

1. Selection of algal species for further study and determination of the optimal

conditions for drying the brown algae to yield high bioactive compound levels.

2. Optimisation of the extraction conditions; isolation and identification of bioactive

compounds from the algal extracts

3. Assessment of the cytotoxic activities of the algal compounds against pancreatic

cancer cell lines in vitro 25

1.3 Research Aims and Expected Outcomes

Aim 1: To select the research objects from six algal species.

Expected outcomes: The physico-chemical profile and antioxidant activities of the extracts will be investigated. Whole and fractionated extracts assessed for bioactivity and antioxidant activities in further studies.

Aim 2: To assess the effects of the drying conditions on the physico-chemical profile and antioxidant activity of the one of six algal species.

Expected outcomes: Optimal drying conditions for high yields of bioactive compounds and antioxidant activities will be identified.

Aim 3: To optimise extraction conditions for high yields of phenolics and high antioxidant activities.

Expected outcomes: The effects of solvents, methods (UAE and MAE) and extraction conditions (temperature, time, power) are evaluated and the optimal extraction parameters for the yields of phenolics and antioxidant activities of the extracts are determined.

Aim 4: Isolation and purification processes are conducted to produce the active individual, or groups of compounds.

Expected outcomes: Individual or groups of compounds with high antioxidant and cytotoxic activities compared to the crude extracts are separated and purified.

Aim 5: To assess cytotoxic activity of the individual compounds, fractions and crude extracts against pancreatic cancer cell lines in vitro.

26

Expected outcomes: Extracts, fractions or individual compounds will be expected to have selective cytotoxic activity against pancreatic cancer cell lines.

1.4 Experimental Rationale

As presented in the Section 1.1, brown algae are a rich source of bioactive compounds, including pigments (carotenes, chlorophyll, fucoxanthin…), phenolic compounds

(phlorotannins), sulfated polysaccharides (fucoidans) and other secondary metabolites.

These compounds have been demonstrated to possess health benefits and a positive link to the treatment of some chronic diseases, including some types of cancer.

To date, little information related to the impact of the drying process on the physicochemical profile and biological activities of brown algae has been reported.

Therefore, it is necessary to evaluate the effect of drying methods (sun-drying, microwave, oven, vacuum, de-humidification and freeze drying) and drying conditions (low and high temperature, sun and microwave irradiation, low pressure, low air humidity) on brown algal components.

Extraction conditions affect the recovery yields and biological activities of plant extracts. A range of extraction solvents should be investigated to assess their impact on the quantity and quality of the extracts and to ensure they do not cause health or environmental concerns.

Optimal extraction methods should enhance extraction efficiency, minimise extraction times and operational costs. To achieve the extraction efficacies of phenolics with high antioxidant activities, the ultrasound and microwave assisted extractions applied with the optimal conditions for brown algae are therefore needed.

Phytochemical profiling of brown algae is a necessary step to select the species that have strong potential for further processing. Therefore, comparisons of the chemical profile

(TPC, TFC, tannins and fucoxanthin content) and antioxidant activities (ABTS, DPPH and

27

FRAP) are evaluated. With the selected species, the isolation of active individual or groups of compounds are required. It is vital to assess potential applications of the extracts by comparison of the biological activity of individual or groups of compounds within extracts.

The isolation of active compounds is required for almost all extracts having further applications in the food, functional food and/or pharmaceutical industries.

Finally, antioxidant assays are a basic assessment for the active individual (fucoxanthin) or groups of algal compounds (phenolics and polysaccharides). The potential applications of algal components will be clearer as the assessments of these components against cancers in vitro and/or in vivo are conducted. Therefore, fucoxanthin, phlorotannins and polysaccharides isolated from brown algae are evaluated against several pancreatic cancer cell lines in vitro.

1.5 Hypothesis, aims and objectives

Importantly, in this study, three main component extracts (fucoxanthin, phenolics and polysaccharides) from brown algae were isolated by several processes (drying, extraction and isolation). Ethanol and water, the two solvents were used for extraction, are environmentally friendly. The algal extracts obtained using these solvent exhibited high antioxidant and cytotoxic activities against pancreatic cancer cell lines.

The hypothesis was that the yields, chemical profile and biological activities of algal compounds could be affected by the sample preparation and extraction method (drying, extraction and isolation), and that enriched extracts and isolated compounds demonstrated potent antioxidant and cytotoxic activity.

Therefore, the overall aims of the study were:

x To select research objects from six algal species

28

x To select optimal drying conditions for each algal sample preparation.

x To optimise yields and antioxidant activities of phenolics for each extraction process.

x To isolate and enrich compounds from isolated crude extracts and assess the cytotoxic

of algal compounds against pancreatic cancer cell lines.

Based on the hypothesis and main aims, the specific objectives of the study were as follows:

1. Paper I: To select the extracts obtained from six brown algae for further

investigation of phytochemical profile through comparison the chemical profile

(TPC, TFC, tannins and fucoxanthin content) and antioxidant activities (ABTS,

DPPH and FRAP) of the extracts.

2. Paper II: Comprehensive evaluation of six drying methods (sun, microwave, oven,

vacuum, de-humidification and freeze drying) and conditions (temperature, time, air

humidity, low pressure) on extract yield, phytochemical profile and antioxidant

activities of algal extracts.

3. Papers III and IV: To optimise the yield of phenolics and antioxidant activities

from H.banksii using ultrasonic water bath (Paper III) and from S.vestitum using

microwave irradiation (Paper IV).

4. Paper V: To investigate the chemical profile and antioxidant activities of the

phenolic-enriched fractions by partition technique from the H.banksii extract.

5. Paper VI: To evaluate the cytotoxic activities of the phenolic-enriched fractions

from the H.banksii extract against several cancer cell lines (via the MTT assay) and

pancreatic cancer cell lines (via the CCK8 assay) in vitro.

29

6. Paper VII: To isolate and purify fucoxanthin from the H.banksii extract by column

chromatography and assess its cytotoxic activity against pancreatic cancer cell lines

in vitro.

7. Paper VIII: To isolate polysaccharide fractions from the H.banksii extract by

precipitation using pure ethanol and centrifugation; and to assess their cytotoxic

activity against pancreatic cancer cell lines in vitro.

The first five papers (I–V) and paper VIII indicated the preparative processes of algal compounds (fucoxanthin, phenolic and polysaccharide fractions) from two brown algae

H.banksii and S.vestitum.

The last three papers (VI–VIII) evaluated the cytotoxic effects of individual compounds

(fucoxanthin), groups of compounds (the phenolic and polysaccharide fractions) and the crude extracts against several cancer cell lines (focusing on pancreatic cancer), in vitro.

30

PART 2: RESULTS

2.1 Synopsis of research papers published from results

In this thesis, the results are presented in a series of eight research papers, four of which

(Papers I to IV) have been published as peer reviewed journal articles, four of which have been submitted for publication.

Paper I, entitled “Comparison of chemical profile and antioxidant properties of the brown algae” evaluated the chemical profile (TPC, TFC, tannins and fucoxanthin content) and antioxidant activities (ABTS, DPPH and FRAP) of six algal extracts, including those from Sargassum vestitum; Sargassum linearifolium; Phyllospora comosa; Padina sp.;

Hormosira banksii and Sargassum podocanthum.

The results showed that the highest TPC (158.85 mg GAE.g-1) and TFC (29.31 mg CAE.g-1) values were recorded for H. banksii, while Padina sp. possessed the largest proportion of tannins (56.17 mg CAE.g-1). The H. banksii, S. vestitum and Padina sp. extracts possessed significantly higher antioxidant activities (ABTS, DPPH and FRAP) compared to the other species examined (P<0.05) and to positive controls: butylated hydroxytoluene, ascorbic acid and D-tocopherol at the concentrations (0.06–1 mg.mL-1). S. vestitum and H. banksii showed high antioxidant activities in the ABTS and DPPH assays, however all extracts generally recorded low FRAP values at all concentrations examined. In addition, fucoxanthin was identified in all six species (0.28–1.97 mg Fx.g-1) by thin layer chromatography (TLC) analysis, was quantified via UV-visible spectrophotometry and isolated for evaluating antioxidant activities. Phenolic compounds were demonstrated to be mainly responsible for antioxidant activities of the extracts, while fucoxanthin also showed relatively high activity.

31

The second research paper entitled “The effects of drying on physico-chemical properties and antioxidant capacity of the brown alga Hormosira banksii (Turner) Decaisne”

(Paper II) evaluated the effectiveness of six drying methods (sun-drying, oven-drying, microwave-drying, vacuum-drying, dehumidification-drying, and freeze-drying) in the temperature range 40–70 °C with reference to impact on physicochemical properties

(recovery and extraction yields, residual moisture, TPC-total phenolic content, TFC-total flavonoid content and proanthocyanidins (tanins) and antioxidant activities (ABTS, DPPH and FRAP) of the H. banksii extract.

The study revealed that mode of drying significantly affected recovery yield, residual moisture, extraction yield, chemical profiles as well as antioxidant capacity of H. banksii.

Phenolics were found to be a main contributor to antioxidant activity of the extracts.

Optimal drying temperatures for oven drying, vacuum drying and de-humidification drying were 40 °C, 50 °C, and 50 °C, respectively, while optimal power for microwave drying was

840 watts. The H. banksii extracts prepared from freeze drying, de-humidification drying and vacuum drying possessed significantly higher levels of TPC, TFC and tannins as well as antioxidant activities (ABTS, DPPH and FRAP) in comparison to extracts derived from samples dried by sun, microwave and oven. Freeze drying was the best for phenolics yield and antioxidant activitiy. As freeze drying is costly and time consuming, de-humidification drying (50 °C; air humidity in and out of 11.1% and 15.4%) and vacuum drying (50 °C, pressure of 10 psi ~ 0.70 kg/cm2~ 517.15 mm Hg) were recommended for the drying of H. banksii.

The third research paper entitled “Optimisation of ultrasound-assisted extraction conditions for phenolic content and antioxidant activities of the alga Hormosira banksii using response surface methodology” (Paper III) estimated the optimal ultrasound-assisted

32 extraction (UAE) conditions for the extraction of phenolics and antioxidant activities from the brown alga H. banksii using response surface methodology (RSM). Box–Behnken design was applied to assess the effect of ultrasonic temperature (30–50 °C), time (20–60 min) and power (60–100% or 150–250 watts) on the total phenolic content (TPC) and antioxidant activity of the extracts.

The results showed that RSM was an accurate and reliable method for estimating extract

TPC level and antioxidant activity (ABTS, DPPH and FRAP) with R2 values of 0.97; 0.96;

0.92 and 0.94, respectively. Temperature and time were found to have a significant impact on TPC level and antioxidant activities. A low temperature (30 °C), modest power (60%,

150 W), and long extraction time (60 min.) were optimal conditions for the phenolic extraction and maximization of antioxidant activities by ABTS and FRAP assay. The best parameters for the DPPH assay were low temperature (30 °C) and power (60% 150 W), and medium time (46.15 min). TPC and antioxidant activities (ABTS, DPPH, FRAP) achieved under the optimal parameters were 23.12 (mg GAE.g-1), 85.64 (mg TE.g-1), 47.24 (mg TE.g-

1) and 12.56 (mg TE.g-1), respectively. UAE was found to be significantly more effective than conventional low temperature extraction (using a shaking water bath at 30 °C for 12h).

The fourth research paper (Paper IV) entitled “Optimum conditions of microwave assisted extraction for phenolic compounds and antioxidant capacity of the brown alga

Sargassum vestitum” aimed to optimise microwave assisted extraction (MAE) conditions for the total phenolic compounds (TPC) and antioxidant activities (ABTS, DPPH and FRAP) from the brown alga S. vestitum using response surface methodology with a Box–Behnken design. The extraction optimisation process utilised a microwave oven (Sharp Caurousel

Inverter microwave 1200W, frequency 2450 MHz, Japan) with an irradiation time range of

33

25–75 seconds, aqueous ethanol solvent percentages (30 – 70%) and microwave power (60

– 100%).

The preliminary experiments of several solvents revealed acetone to be the most effective for extraction of phenolics, recording the highest values of yield and antioxidant activities compared to other organic solvents including methanol, ethanol, and ethyl acetate (Paper

IV). However, acetone is less cost effective than other solvents, and acetone residue is not desirable for food and pharmaceutical applications when compared to solvents such as ethanol. Therefore, ethanol is a suitable solvent for extraction. Ethanol concentration had a significant impact both phenolic yield and antioxidant activities of the extracts, as did irradiation time and microwave power. Optimal MAE conditions were determined as: ethanol concentration (70%), irradiation time (75s) and microwave power (80% = 960W).

Under optimal conditions, the maximum values of TPC and antioxidant activities (ABTS,

DPPH and FRAP) achieved were 58.2 mg GAE.g-1, 149.81 mg TE.g-1, 116.54 mg TE.g-1,

67.95 mg TE.g-1, respectively. In comparison with the conventional extraction (temperature of 30 °C, shaking water bath for 12h) and ultrasonic extraction (ultrasonic temperature of 30

°C, time of 60 min and power of 60%), the optimal MAE extraction efficacy proved to be the most efficient. The VWXG\ FRQILUPHG 0$( XVLQJ 560 ZDV D UHOLDEOH DQG HI¿FLHQW method for extraction of the high yields of phenolics in the alga S.vestitum

The fifth research paper (Paper V) entitled “Chemical profile and antioxidant activities of the crude extract and different fractions prepared from the brown alga

Hormosira banksii (Turner) Decaisne” aimed to determine chemical and antioxidant properties of the crude extract and its fractions prepared from the brown alga H. banksii by the solvent-partition technique.

34

The results showed that the organic fractions: ethyl acetate (EA), butanol (BuOH), hexane

(Hx) and dichloromethane (DCM) had significantly higher levels of phenolics, flavonoids and tannins, while components from the aqueous fraction (AQ) were lower than those of the crude extract (CE). There was a strong correlation between compounds and antioxidant activities of the extract (r2= 0.63–0.99). Among the organic fractions, the EA fraction possessed the highest concentration of phenolics and antioxidant activities, with the latter comparable to the positive controls, such as butylated hydroxytoluene (BHT), L-ascorbic

DFLGDQGĮ-tocopherol at concentrations of 0.06–1.00 mg.mL-1. It is suggested that solvent- partition method was efficient in separating compounds with different polarity within the algae. Phenolic compounds (phlorotannins) were the main contributors to the antioxidant activity of the extract.

The sixth research paper (Paper VI) entitled “Fucoxanthin content, isolation and cytotoxic activity against pancreatic cancer from brown alga Hormosira banksii (Turner)

Decaisne” was undertaken to isolate, purify and assess the cytotoxic activity of fucoxanthin against pancreatic cell lines. HPLC analysis showed the fucoxanthin content isolated from the alga to be 0.58 mg Fx.g-1 alga, with purity estimated at 92.3% using column chromatography. Fucoxanthin showed high anti-proliferative activity (30.91– 92.81%) on both primary (MiaPaCa-2) and secondary (BxPC3, CFPAC1) pancreatic cancer cell lines at concentrations of 100-200 μg.mL-1 with medium toxicity against non-tumorigenic (HPDE)

-1 cells (IC50 value of 71.81 mg.mL ). The study provides a rationale for further assessment of fucoxanthin’s mode of action in the cell cycle.

The seventh research paper (Paper VII) entitled “Antioxidant and cytotoxic activity

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

(Turner) Decaisne” aimed to evaluate the antioxidant and cytotoxic activity of the

35 phlorotannin-enriched fractions from the brown algae Hormosira banksii on pancreatic cancer cell lines (MiaCaPa-2, BxPC3 and CFPAC1) as well normal pancreatic epithelial cells (HPDE).

The results revealed that the phlorotannin-enriched fractions (ethyl acetate (EA) and butanol fraction (BuOH)) had high antioxidant activity, and strongly inhibited cancer cell growth.

-1 The EA fraction (IC50 = 29.08 μg.mL ) suppressed MiaCaPa-2 cell growth by 79.42% at the low concentration of 50 μg.mL-1, while the BuOH fraction showed high growth inhibition of 79.14% at 100 μg.mL-1. However, the medium-polar phenolics (EA fraction) were found to be highly toxicity to the normal cells (HPDE) with an IC50 value of 1.72

μg.mL-1. Interestingly, the polar phenolics (BuOH fraction) showed selective anticancer

-1 activity with an IC50 value of 106.45 μg.mL for the normal cells. Therefore, phlorotannin- enriched fractions showed impressive growth inhibition efficacy against pancreatic cancer cell lines and further investigations should be conducted for delineation of their effects on apoptotic and cell cycle mechanisms.

The eighth research paper (Paper VIII) entitled “Extraction and cytotoxic activity of polysaccharides (fucoidan) from brown alga Hormosira banksii (Turner) Decaisne” evaluated the chemical profile, antioxidant and cytotoxic activities of the polysaccharide fractions (CF50, CF70 and CFR) against pancreatic cancer cell lines.

The results revealed that the highest yield was observed in the CF50 fraction (30.53%), followed by the CF70 fraction (9.56%) and then the CFR fraction with only 5.82% (% dry weight). The total sugars were significantly different among the fractions. All the polysaccharide fractions (CF50, CF70 and CFR) were fucoidans due to the presence of sulfate content with 10.59%; 12.19% and 11.00%, respectively. Antioxidant activities (the

ABTS, DPPH and FRAP assays) of these polysaccharide fractions were weak. However,

36 these fractions were shown to possess high cytotoxic activities (the inhibition of 39.35 –

82.82% at the concentrations of 100 – 200 μg.mL-1) against pancreatic cancer cell lines

(MiaCaPa-2, BxPC3 and CFPAC1) in a dose-dependent manner. In addition, the CF50 and

CF70 fractions possessed selective cytotoxicity due to low toxicity to the non-tumourigenic

-1 cells (HPDE) with the IC50 values of 526.32 and 781.25 μg.mL , respectively.

2.2 Research papers published from results

The results for the current thesis are based on the following eight research result papers:

2.2.1 Paper I: Dang TT, Bowyer MC, Van Altena IA & Scarlett CJ. (2017). Comparison of

chemical profile and antioxidant properties of the brown algae. International Journal

of Food Science & Technology 51(1): 174-181. doi: 10.1111/ijfs.13571.

2.2.2 Paper II: Dang TT, Vuong QV, Schreider MJ, Bowyer MC, Van Altena IA &

Scarlett CJ. (2017). The Effects of Drying on Physico-Chemical Properties and

Antioxidant Capacity of the Brown Alga (Hormosira banksii (Turner) Decaisne).

Journal of Food Processing and Preservation 41(4): e13025.

doi.org/10.1111/jfpp.13025.

2.2.3 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. Journal of Applied Phycology 29(6): 3161-3173.

doi.org/10.1007/s10811-017-1162-y.

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

conditions of microwave assisted extraction for phenolic compounds and antioxidant

capacity of the brown alga Sargassum vestitum. Separation Science and Technology

(In Press). doi.org/10.1080/01496395.2017.1414845.

37

2.2.5 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.

2.2.6 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.

2.2.7 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 Marine

Biotechnology.

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

polysaccharides (fucoidans) from brown alga Hormosira banksii (Turner) Decaisne.

Submitted to Journal of Biomedicine and Pharmaotherapy.

38

Paper I:

“Comparison of chemical profile and antioxidant properties of the brown algae”. International Journal of Food Science & Technology 51(1): 174-181. doi: 10.1111/ijfs.13571.

174 International Journal of Food Science and Technology 2018, 53, 174–181

Original article Comparison of chemical profile and antioxidant properties of the brown algae

Thanh T. Dang,1,2 Michael C. Bowyer,1 Ian A. Van Altena1 & Christopher J. Scarlett1*

1 School of Environmental and Life Sciences, Faculty of Science, University of Newcastle, Brush Rd, Ourimbah, NSW 2258, Australia 2 Department of Seafood Processing Technology, Faculty of Food Technology, Nha Trang University, 02 Nguyen Dinh Chieu Street, Nha Trang City, Khanh Hoa, Vietnam (Received 27 April 2017; Accepted in revised form 26 July 2017)

Summary Six brown algae, Sargassum vestitum, Sargassum linearifolium, Phyllospora comosa, Padina sp., Hormosira banksii and Sargassum podocanthum, were investigated for the chemical profile and antioxidant activity. The results showed that the extracts H. banksii, S. vestitum and Padina sp. indicated the significantly higher total phenolic compound (TPC) and antioxidant activities (ABTS, DPPH and FRAP) compared to the other species (P<0.05) and comparable to positive controls: butylated hydroxytoluene, ascorbic acid and alpha-tocopherol at the concentrations (0.06–1mgmL1). Fucoxanthin was also found in six species and isolated for evaluating antioxidant activity. In addition, the phenolic compounds were mainly respon- sible for antioxidant activity of the extracts, while fucoxanthin showed quite high antioxidant activity. It is suggested that S. vestitum, H. banksii and Padina sp. have the potent for extracting bioactive compo- nents and further applications in food and pharmaceutical industries.

Keywords Algae, antioxidant activity, extraction/separation, phenols, pigments.

exist in these harsh conditions (Jimenez-Escrig et al., Introduction 2001). On the other hand, the role of the natural In recent years, marine algae have been increasingly antioxidants from algae has been increasing in food or attracted in the search for bioactive compounds to medicinal materials because the synthetic antioxidants develop new drugs and healthy foods (Mohamed (butylated hydroxyanisole (BHA), butylated hydroxy- et al., 2012). Particularly, brown algae (Phaeophyta) toluene (BHT) and alpha-tocopherol) used in reducing are of great interest due to their potential ability to oxidative damages need to replace due to side effects produce a variety of secondary metabolites such as such as liver damage, carcinogenesis (Munir et al., fucoxanthin, phenolic compounds, sulphated polysac- 2013). Brown algae have been reported to have compar- charide, terpenoids, bromophenols which can benefit atively higher antioxidant potential than green and red human health (Gupta & Abu-Ghannam, 2011; Balboa algae (Balboa et al., 2013). In brown algae, phenolic et al., 2013). These components have been found to compounds have a high ratio compared to the others exhibit anticancer activity against several types of can- and are mainly responsible for antioxidant activity of cers in vitro (Dellai et al., 2013; Kumar et al., 2013). the extracts (Wang et al., 2009; Cox et al., 2010; Farvin In the marine environment, algae are exposed to & Jacobsen, 2013). The structure of these compounds extreme conditions (ultraviolet light, salinity, tempera- varies from the simple molecules, such as phenolic acid, ture and high oxygen concentrations), leading to the to more complex compounds constructed by several formation of free radicals and other oxidising agents. units of the phloroglucinol monomer (1, 3, 5-trihydrox- However, they still developed well without any serious ybenzene) called phlorotannins (Gupta & Abu-Ghan- structural and photodynamic damage during metabo- nam, 2011). Besides, pigment such as fucoxanthin is lism. From this fact, it can be suggested that they pos- one of the most abundant carotenoids of brown algae sessed some protective mechanisms and/or produced and estimates around 10% of total carotenoids found several secondary metabolites which helped them to in nature (Rajauria et al., 2016). The structure of fucoxanthin includes a usual allenic bond and 5, 6- monoepoxide in its molecule, and almost all of them *Correspondent: Fax: +61 2 4348 4145; e-mail: [email protected] were trans-fucoxanthin in brown algae (Jaswir et al.,

doi:10.1111/ijfs.13571 © 2017 Institute of Food Science and Technology Comparison of antioxidant activity of algae T. T. Dang et al. 175

2013). Fucoxanthin was also responsible for antioxi- cations. The absorbance was measured at 765 nm dant activity of algal extracts reported by several using a UV spectrophotometer (Varian Australia Pty. researchers (Fung et al., 2013; Foo et al., 2015). Ltd., Bundoora, Vic., Australia). Gallic acid was used The reports relevant to the properties of brown as a standard and the results were expressed as mg of algae Sargassum vestitum, Sargassum linearifolium, gallic acid equivalents per gram of the extract (mg Phyllospora comosa, Padina sp., Hormosira banksii and GAE g1 extract). Sargassum podocanthum in eastern coast of Australia Total flavonoid content (TFC) was measured as are limited. Therefore, this study aimed to investigate described by Zhishen et al. (1999). The absorbance the chemical profile and antioxidant activity of six was measured at 510 nm. Catechin was used as a stan- algal species. Fucoxanthin purified from Hormosira dard and the results were expressed as mg of catechin banksii was also evaluated for its contribution to equivalents per gram of the extract (mg CAE g1 antioxidant activity compared to phenolic compounds extract). in the extracts. The conclusion is the important evi- Total condensed tannin content (Tannins) was deter- dence for further isolation and purification of the mined as described by Cox et al. (2010) with slight bioactive compounds. modifications. The absorbance was measured at 500 nm. Catechin was used as the standard and the results were expressed as mg CAE g1 extract. Material and methods

Chemicals Fucoxanthin content of the extracts and thin-layer chromatography (TLC) All chemicals for assays and the standard fucoxanthin (95%) were purchased from Sigma Chem. Co. (Castle Fucoxanthin content was quantified by reading the Hill, NSW, Australia). absorbance at their respective wavelengths through UV–visible spectrophotometer (Varian 300) and using the formulae given below by Seely et al. (1972) and Algal samples Sudhakar et al. (2013). Six brown algae, S. vestitum, S. linearifolium, P. co- ÀÁ mosa, Padina sp., H. banksii and S. podocanthum were Fucoxanthin mg g 1 ¼ collected from the Bateau Bay, NSW, Australia, in March, 2016. These algal species were identified by A470 1:239ðÞA631þA581 0:3 A664 Dr. Maria Schreider, School of Environmental and 0:0275 A664=141 Life Sciences, University of Newcastle. After collec- = tion, the samples were washed with sea water to where A470, A581, A631, A664 absorbance at the differ- remove natural residues (sand, salt and epifauna) and ent wavelengths of 470, 581, 631 and 664 nm, respec- washed thoroughly with fresh water before drying. tively. The results were expressed as mg of fucoxanthin per gram of dried alga (mg Fx g1 alga). Fucoxanthin from alga H. banksii was isolated, puri- Preparation of the algal extracts fied using partition, column chromatography and iden- The samples were freeze-dried for 48 h using a freeze tified by comparing with a standard fucoxanthin (95%). dryer and the dried algae were ground to give ≤0.6 mm This compound was evaluated the antioxidant activity 1 particle size. The algae were extracted with the ethanol at the serial concentrations (0.03–1mgFxmL ). TLC (70%) using ultrasonic bath (Soniclean, 220 V, 50 Hz for the extracts was performed on a silica gel plate 9 and 250W; Soniclean Pty Ltd, Stepney, SA, Australia) (20 20 cm, Kieselgel 60F254, 0.25 mm, Merck, Castle set at temperature of 30 °C, time of 60 min and power Hill, NSW, Australia). The mixture of hexane and ace- of 150W as described by Dang et al. (2017). The process tone with the ratio of 7:3 (v/v) was used as the mobile was repeated (n = 3) till the samples became colourless. phase. The extracts were filtered and the filtrates were concen- trated using a rotary evaporator (Buchi Rotavapor Antioxidant activity of the algal extracts B-480, Noble Park, Vic., Australia). These extracts were freeze-dried to obtain the crude powders and then Three different antioxidant assays were performed for stored at 20 °C until analysis. the extracts and the positive controls: butylated hydroxytoluene (BHT) (99% purity), L-ascorbic acid (95% purity) and alpha-tocopherol (90% purity) at Chemical properties of the algal extracts the concentrations of 0.06–1mgmL 1. Total phenolic content (TPC) was determined as ABTS total antioxidant capacity (ABTS) was mea- described by Wang et al. (2009) with slight modifi- sured based on the method described by Foo et al.

© 2017 Institute of Food Science and Technology International Journal of Food Science and Technology 2018 176 Comparison of antioxidant activity of algae T. T. Dang et al.

(2015). The absorbance was taken at 734 nm using a values were found in the extracts Laminaria digitata, spectrophotometer. Trolox was used as a stan- Laminaria saccharina and Himanthalia elongata dard and the results were expressed as mg TE g1 (37.66–151.33 mg GAE g1 extract). There was a wide extract. range of TPC reported in the different Icelandic sea- DPPH radical scavenging activity (DPPH) was mea- weeds between 4 and 242 mg PGE g1 extract (Wang sured based on the method described by Brand-Wil- et al., 2009). However, the low TPC levels of brown liams et al. (1995) with slight modifications. The seaweed extracts (24.61 and 49.16 mg GAE g1 samples were read at 515 nm and the results were extract) were also outlined by Chandini et al. (2008). expressed as mg TE g1 extract. It is clear that different species resulted in the differ- Ferric reducing antioxidant power (FRAP) was con- ence in phenolic compounds of the extracts (Cho ducted according to Benzie & Strain (1999). Readings et al., 2010). Moreover, it was not only the diversity of of the samples were taken at 593 nm and results were the algal species, but also the environment conditions expressed as mg TE g1 extract. and locations affected amount of TPC in the algae (Jimenez-Escrig et al., 2001). Flavonoids have been reported as antioxidants, Statistical analysis scavengers of a wide range of reactive oxygen species All experiments were performed at least in triplicate. (Zhishen et al., 1999). It is known that the flavonoids, The data were expressed as mean standard devia- a large group of phenolic compounds as flavonols, fla- tion (n = 3). A one-way ANOVA and LSD post hoc test vones, catechins, proanthocyanidins, anthocyanidins were employed (SPSS Statistical Software, version 16, and isoflavonoids have a broad spectrum of the bio- SPSS Inc., Chicago, IL, USA) to analyse the differ- logical activities (Kahk€ onen€ et al., 1999; Cox et al., ences between the algal species. Differences between 2010). Among the brown algae, it was found that the the mean levels of the components and antioxidant TFC of H. banksii was the highest (29.31 mg CAE g1 activities in different experiments were taken to be sta- extract). Sargassum podocanthum and Padina sp. also tistically significant at P < 0.05. had the high TFC values with 22.38 and 20.74 mg CAE g1 extract, respectively. The lowest TFC was of 9.89 mg CAE g1 extract observed at the extract Results and discussion P. comosa. Cox et al. (2010) outlined that there was a wide range of TFC from the brown, red and green Chemical properties of the algal extracts algae (7.66 and 42.5 mg QE g 1 extract) and the The TPC values of the six species are presented in brown alga H. elongata had the highest TFC com- Table 1. Among the algae, TPC of H. banksii was the pared to the other species. In addition, flavonoids highest at 158.82 mg GAE g1 extract and there was could be mainly responsible for antioxidant and no significant difference in TPC values between antimicrobial activities of the algal extracts. The high extracts S. linearifolium and S. podocanthum. Three values of TFC were also found in brown algae Padina algae S. vestitum, H. banksii and Padina sp. displayed gymnospora (Kutzing)€ Sonder and Sargassum wightii significantly higher TPC compared to the rest of the Greville ex J. Agardh compared to red and green algae investigated species (P<0.05, Table 1). It was in line using different solvents for extraction (Murugan & with Cox et al. (2010) who stated that the high TPC Iyer, 2013). Tannins are known as the secondary metabolites including condensed compounds (gallic and/or egallic Table 1 Chemical profiles of the six algal extracts. Values are acid) and hydrolysable compounds (polymeric flavo- shown as the mean standard deviation (n = 3) and the different noids) which are widespread among terrestrial and letters in the same column are significantly different (P < 0.05). The 1 marine plants (Huang et al., 2008). From the findings, values were expressed as mg per gram of the extract (mg g extract) there was a significant difference in tannins of the extracts (P < 0.05, Table 1). It was shown that the TPC TFC Tannins 1 Algae species (mg GAE g1) (mg CAE g1) (mg CAE g1) highest tannins was in Padina sp. (56.17 mg CAE g extract), followed by H. banksii (45.91 mg CAE g1 Sargassum vestitum 141.91 3.95a 17.78 0.74a 24.39 0.37a extract) and then S. podocanthum (39.07 mg Sargassum 47.06 0.65b 13.93 0.41b 17.83 0.41b CAE g1 extract). The lowest value was in S. lineari- linearifolium folium (17.83 mg CAE g1). The results were in line c c c Phyllospora comosa 67.78 1.01 9.89 0.41 20.98 0.44 with Cox et al. (2010) who revealed that the content Padina sp. 124.65 0.78d 20.74 0.49d 56.17 0.22d e e e of tannins from the edible Irish algae ranged between Hormosira banksii 158.82 1.19 29.31 0.88 45.91 0.87 1 Sargassum 48.13 0.66b 22.38 0.43f 39.07 2.34f 3.19 and 38.34 mg CAE g extract. Total condensed podocanthum tannin content in the extracts of H. elongata ranged between 9.8 and 35.6 mg CAE g1 extract with

International Journal of Food Science and Technology 2018 © 2017 Institute of Food Science and Technology Comparison of antioxidant activity of algae T. T. Dang et al. 177 different ratios of methanol and water (Rajauria et al., Sargassum wightii, Sargassum ilicifolium, Sargassum 2013). In comparison with other brown algae reported, longifolium, Padina gymnospora and Turbinaria ornata at three algae S. vestitum, H. banksi and Padina sp. 0.03–0.38 mg Fx g1 alga (Sudhakar et al., 2013). The showed the high levels of TPC, TFC and tannins. low amount of fucoxanthin was also outlined in Turbina- Therefore, these algae could be potential rich sources ria turbinata, and Sargassum plagyophyllum with 0.59 of natural antioxidants for protection of food against and 0.71 mg Fx g1 alga, respectively (Jaswir et al., oxidation and further applications in pharmaceutical 2013). In comparison with macroalgae, Foo et al. (2017) fields. showed that microalgae (Chaetoceros calcitrans with 5.13 mg Fx g1 alga) had higher fucoxanthin compared to macroalgae, and it could be up to 18.23 mg Fx g1 Fucoxanthin content and thin-layer chromatography alga in microalgae Isochrysis aff. galbana (Kim et al., (TLC) 2012). The fucoxanthin content of the algal species was calcu- In TLC profiles, the pigment (orange yellow band) lated through the formula described by Seely et al. isolated from H. banksii was confirmed to be fucoxan- (1972) and visualised through TLC plate (Table 5 and thin by comparing to the standard fucoxanthin on Fig. 1a–c). Fucoxanthin was the highest in Padina sp. TLC plate (Fig. 1c). Figure 1a shows the fucoxanthin (1.97 mg Fx g1 alga). Sargassum linearifolium, S. vesti- content of six species. The strong or light yellow bands tum and S. podocanthum also had high ratio of this of the species responded to different amount of fucox- compound at 1.76; 1.65 and 1.46 mg Fx g1 alga, anthin in the extracts. In Fig. 1b, fucoxanthin was respectively. The low amount of this pigment was found shown in the nonpolar solvent fractions (hexane and in H. banksii and P. comosa (0.61 and 0.28 mg Fx g1 dichloromethane fraction) through partition. alga, respectively). The results were supported by Tera- saki et al. (2009) who outlined that fucoxanthin had a Antioxidant activities of the algal extracts wide range in brown algae from 0.1 mg Fx g 1 alga (Desmarestia viridis) to 3.7 mg Fx g1 alga (Sargassum ABTS assay based on the scavenging ability of antioxi- horneri). Fucoxanthin content of five out of six species in dants to the radical anion ABTS*+ was used to deter- this study was higher than that of five brown algae mine antioxidant activity of six algal extracts. The

(a) (b) (c)

Figure 1 (a) TLC for fucoxanthin of the six species (S1, S2, S3, S4, S5 and S6 represent Sargassum vestitum, Sargassum linearifolium, Phyl- lospora comosa, Padina sp., Hormosira banksii and Sargassum podocanthum, respectively); (b) TLC for fractions of H. banksii (F1, F2, F3, F4 and F5 represent hexane, dichloromethane, ethyl acetate, butanol and aqueous fraction, respectively). Fucoxanthin in band 6 (B6) was isolated and determined the antioxidant activity; (c) TLC of fucoxanthin (Fuco) purified from H. banksii compared to a standard fucoxanthin (Std). [Colour figure can be viewed at wileyonlinelibrary.com]

© 2017 Institute of Food Science and Technology International Journal of Food Science and Technology 2018 178 Comparison of antioxidant activity of algae T. T. Dang et al.

Table 2 Antioxidant activity (ABTS) of six algal extracts and positive controls at the different concentrations. The value was expressed as mg trolox equivalent per gram the extract (mg TE g1 extract). Butylated hydroxytoluene (BHT), ascorbic acid (A.A), alpha-tocopherol (Toco.). The different letters in the same column are significantly different (P < 0.05)

ABTS (mg TE g1 extract)

Algae species 0.06 0.12 0.25 0.50 1.00 (mg mL1)

Sargassum vestitum 31.71 1.77a 65.77 0.79a 114.48 2.25a 170.53 1.46a 183.38 0.05a Sargassum linearifolium 2.02 0.51b 3.76 0.61b 9.42 0.37b 16.42 1.32b 34.52 1.92b Phyllospora comosa 9.70 0.24c 17.78 0.42c 34.29 0.39c 65.72 1.67c 117.41 3.04c Padina sp. 22.53 0.88d 46.65 2.41d 81.80 2.86d 139.64 2.08d 183.71 0.22a H. banksia 45.87 0.77e 80.96 1.84e 135.77 2.91e 167.44 0.21e 167.90 0.12d Sargassum podocanthum 13.30 0.69f 24.18 0.77f 47.85 0.21f 92.31 0.82f 147.09 1.96e BHT 76.89 2.07g 132.42 4.62g 160.88 0.73g 164.20 0.11g 164.41 0.39f A.A 104.27 6.10h 165.16 0.08h 165.22 0.17h 165.30 0.12h 165.35 0.09g Toco. 45.14 2.20e 97.25 3.53i 163.95 0.51i 165.77 0.04i 165.84 0.04h

ABTS values of the extracts were investigated and In regard to fucoxanthin, it was shown that ABTS compared with the synthetic antioxidants (BHT, ascor- of fucoxanthin ranged from 3.06 to 102.63 mg TE g1 bic acid and alpha-tocopherol). It was found that the fucoxanthin at the concentrations 0.03–1mgFxmL1 antioxidant activities of all extracts were dose-depen- (Table 5). It can be seen that fucoxanthin had the dent in the range of the tested concentrations. ABTS quite high antioxidant activity. However, its activity levels of the extracts H. banksii; S. vestitum and Pad- was lower compared to extracts and positive controls. ina sp. were significantly higher in comparison with The ratio of fucoxanthin was also lower than phenolic those of other species at all concentrations (P < 0.05, compounds in the extracts. In addition, H. banksii Table 2). Especially, there was no significant difference with the low fucoxanthin (0.61 mg Fx g1 alga) still in antioxidant activity between H. banksii and alpha- had higher ABTS than Padina sp.; S. linearifolium and tocopherol at the lowest concentration (P>0.05) and S. vestitum with high fucoxanthin content (1.97, 1.76 comparable to alpha-tocopherol at the other concen- and 1.65 mg Fx g1 alga, respectively). Therefore, the trations tested. In addition, ABTS levels of three algae contribution to antioxidant activity of fucoxanthin was H. banksii; S. vestitum and Padina sp. were compara- lower than of phenolic compounds in the extracts. ble to all positive controls (BHT, ascorbic acid and DPPH assay was used for testing the free radical alpha-tocopherol) at the concentrations ≥0.5 mg mL1 scavenging activities of the extracts. Brand-Williams and higher than that reported by the previous studies. et al. (1995) reported that DPPH radical scavenging It was shown that the ABTS value of Bifurcaria bifur- capacity of components depended on their ability to cata (728.29 lmol TE g1) was seven times lower than pair off with the unpaired electron of a radical. The that of BHT (5105.03 lmol TE g1) (Agregan et al., DPPH radical scavenging activities of the extracts are 2016). Foo et al. (2017) reported that the ABTS of the outlined in Table 3. The antioxidant activity of the microalgae Isochrysis galbana (21.55 mg TE g1 alga) extracts significantly differed (P < 0.05), and the and Chaetoceros calcitrans (16.92 mg TE g1 alga) was extract S. vestitum was the most potent scavenger in lower compared to the findings in our study. these algae tested (29.08–209.5 mg TE g1 extract).

Table 3 Antioxidant activity (DPPH) of six algal extracts and positive controls at the different concentrations. The different letters in the same column are significantly different (P < 0.05)

DPPH (mg TE g1 extract)

Algae species 0.06 0.12 0.25 0.50 1.00 (mg mL1)

Sargassum vestitum 29.08 0.80a 56.17 0.99a 97.93 1.36a 158.24 4.74a 209.50 0.52a Sargassum linearifolium 0.11 0.06b 0.43 0.06b 2.86 0.29b 5.98 0.31b 15.57 0.82b Phyllospora comosa 12.35 0.26c 25.86 1.19c 46.69 0.69c 82.76 0.56c 117.25 2.99c Padina sp. 22.42 0.22d 36.99 1.63d 59.54 0.84d 93.73 1.65d 138.15 3.50d H. banksia 28.88 2.72a 48.70 1.18e 80.25 1.51e 130.98 0.86e 177.32 1.12e Sargassum podocanthum 12.93 0.53c 26.26 0.85c 50.35 0.93f 87.08 0.32f 136.59 4.17d BHT 27.89 1.12a 43.70 1.88f 80.19 4.71e 126.57 1.60g 178.40 2.26e A.A 96.26 1.18e 204.55 2.90g 209.54 5.08g 212.49 0.15h 212.49 0.14f Toco. 47.17 1.63f 94.94 4.71h 172.42 5.23h 214.78 0.45i 215.73 0.31g

International Journal of Food Science and Technology 2018 © 2017 Institute of Food Science and Technology Comparison of antioxidant activity of algae T. T. Dang et al. 179

DPPH values of the three algae S. vestitum, H. banksii ferrous complex (Fe2+) by electron transfer, which and Padina sp. were high at the low concentrations. indicates the capacity of the compound to reduce reac- There was no significant difference in antioxidant tive species (Benzie & Strain, 1999). The results from activities between H. banksii and S. vestitum at the Table 4 showed that almost all of the extracts had low lowest concentration (P > 0.05). Interestingly, both FRAP values (1.04–43.09 mg TE g1 extract), while extracts above had DPPH levels significantly higher S. vestitum still indicated the excellent antioxidant than of BHT at all concentrations (P < 0.05). In addi- activity (20.3–283.71 mg TE g1 extract) at the differ- tion, the DPPH values of Padina sp.; P. comosa and ent concentrations. The FRAP value of S. vestitum S. podocanthum were also quite high (≥80 mg TE g1 was even higher than that of BHT at the concentra- extract) at the concentrations ≥0.5 mg mL1. It was in tions ≥0.25 mg mL1. In addition, the positive con- agreement with Farvin & Jacobsen (2013) who trols such as ascorbic acid and alpha-tocopherol had reported that the extracts of Fucus vesiculosus and the high FRAP values (111.17 mg TE g1 and 1 1 Fucus serratus (EC50 9.9 and 9.2 lgmL , respec- 30.15 mg TE g , respectively) even at the lowest con- tively) with high TPC levels had potential DPPH radi- centration (0.06 mg mL1). In terms of fucoxanthin, cal scavengers, even higher than positive control BHT the antioxidant activity for fucoxanthin was also 1 (EC50 10.7 lgmL ). However, Wang et al. (2009) observed (Table 5). It ranged from 2.19 to 43.71 mg revealed that all the positive controls ascorbic acid TE g1 extract when the concentration increased 1 1 1 (EC50 = 2.5 lgmL ), BHT (EC50 = 3.3 lgmL ) between 0.03 and 1 mg mL . 1 and alpha-tocopherol (EC50 = 5.9 lgmL ) exhibited It can be seen that antioxidant activities (ABTS and higher DPPH values compared with algal extracts DPPH) of three algae S. vestitum, H. banksii and Pad- 1 Fucus vesiculosus (EC50 = 10.7 lgmL ), Fucus serra- ina sp. were higher than those of other algae tested. 1 tus (EC50 = 11.0 lgmL ) and Ascophyllum nodosum However, in FRAP assay, it was found that only 1 (EC50 = 18.5 lgmL ). Antioxidant activity of the S. vestitum had high FRAP value compared to that of brown algae shown by Zhang et al. (2007) was much ascorbic acid and alpha-tocopherol. Therefore, it could lower than positive controls ascorbic acid, BHT and be that extract S. vestitum had a quite high rate of gallic acid. ascorbic acid and/or alpha-tocopherol. This was sup- DPPH radical scavenging capacity of fucoxanthin ported by Farvin & Jacobsen (2013) who indicated is presented in Table 5. It was higher than BHT at that brown algae contain higher level of tocopherols the low concentration (≤0.06 mg mL1). However, than red algae and antioxidant activity of the algal the activity of fucoxanthin increased slowly with the extracts increased due to the presence of tocopherols increase of its concentration. It reached to 88.26 mg in the extracts, while ascorbic acid was also found in TE g1 at the concentration of 1 mg mL1. The find- the algae reported by Fayaz et al. (2005). Further- ings were supported by Fung et al. (2013) who showed more, the previous studies also reported that the that fucoxanthin in Undaria pinnatifida had a close FRAP values of Bifurcaria bifurcata at 26.93 lmol correlation with DPPH level of the extract (R2 = 0.88). TE g1 extract (Agregan et al., 2016) and Saccharina Therefore, fucoxanthin content in the algae studied longicruris at 22.3 lmol TE g1 extract (Boisvert et al., also accounted for antioxidant activity of the extracts. 2015) were around 20 times lower in comparison with The FRAP assay measures the ability of an antioxi- FRAP value of BHT (578.50 lmol TE g1). Foo et al. dant compound to reduce a ferric oxidant (Fe3+)toa (2017) indicated the low antioxidant activity of the

Table 4 Antioxidant activity (FRAP) of six algal extracts and positive controls at the different concentrations. The different letters in the same column are significantly different (P < 0.05)

FRAP (mg TE g1 extract)

Algae species 0.06 0.12 0.25 0.50 1.00 (mg mL1)

Sargassum vestitum 20.30 0.49a 43.06 0.09a 84.62 4.75a 166.27 7.86a 283.71 8.38a Sargassum linearifolium 1.04 0.10b 1.52 0.06b 2.46 0.07b 6.55 0.09b 12.52 0.19b Phyllospora comosa 3.52 0.07c 6.39 0.18c 6.80 0.37c 11.04 0.13c 15.91 0.13c Padina sp. 5.10 0.18d 8.86 0.05d 10.11 0.07d 20.84 0.51d 34.53 0.32d H. banksia 4.16 0.71e 8.17 1.83d 12.46 0.90e 22.70 0.82e 43.09 0.90e Sargassum podocanthum 1.06 0.16b 2.70 0.12e 7.55 0.10f 14.12 0.08f 29.64 0.58f BHT 28.53 1.02f 49.64 0.86f 69.82 1.78g 103.97 2.82g 184.51 6.40g A.A 111.17 1.47g 240.69 2.57g 436.34 6.90h 471.54 8.73h 474.91 3.96h Toco. 30.15 0.45h 71.00 1.78h 174.49 7.89i 371.02 8.66i 472.05 4.82h

© 2017 Institute of Food Science and Technology International Journal of Food Science and Technology 2018 180 Comparison of antioxidant activity of algae T. T. Dang et al.

Table 5 The fucoxanthin content (mg Fx g1 alga) in six species and antioxidant activity (mg TE g1 fucoxanthin) of the purified fucoxanthin of Hormosira banksii at different concentrations. The different letters in the same row are significantly different (P < 0.05)

Algae species Sargassum vestitum Sargassum linearifolium Phyllospora comosa Padina sp. H. banksii Sargassum podocanthum

Fucoxanthin 1.65 0.05a 1.76 0.03b 0.28 0.01c 1.97 0.03d 0.61 0.02e 1.46 0.06f

Antioxidant activities of fucoxanthin from H. banksii at different concentrations (mg mL1) Concentration 0.03 0.06 0.12 0.25 0.50 1.00

ABTS 3.06 0.46 7.22 0.39 17.09 1.10 29.77 1.27 59.75 1.82 102.63 2.89 DPPH 22 1.35 30.6 1.28 37.89 1.48 45.65 0.98 60.72 1.27 88.26 1.96 FRAP 2.19 0.11 3.84 0.22 6.82 0.08 12.16 0.27 27.05 1.38 43.71 1.43

microalgae Chaetoceros calcitrans (1.65 mg TE g 1 activity of Bifurcaria bifurcata aqueous extract on canola oil. Effect alga) and Isochrysis galbana (1.96 mg TE g1 alga). of extract concentration on the oxidation stability and volatile compound generation during oil storage. Food Research Interna- The FRAP values for the extracts of Kappaphycus tional, 99, 1095–1102. alvarezzi and Padina antillarum were only 0.56 and Balboa, E.M., Conde, E., Moure, A., Falque, E. & Domınguez, H. 15.7 mg TE g 1 alga, respectively, as observed by (2013). In vitro antioxidant properties of crude extracts and com- Chew et al. (2008). pounds from brown algae. Food Chemistry, 138, 1764–1785. Benzie, I. & Strain, J. (1999). Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological flu- ids and modified version for simultaneous measurement of total Conclusion antioxidant power and ascorbic acid concentration. Methods in Enzymology, 299,15–27. The study indicated that six algal species had the sig- Boisvert, C., Beaulieu, L., Bonnet, C. & Pelletier, E. (2015). nificant difference in terms of TPC, TFC, tannins and Assessment of the antioxidant and antibacterial activities of fucoxanthin as well as antioxidant activities (ABTS, three species of edible seaweeds. Journal of Food Biochemistry, DPPH and FRAP). The algae S. vestitum, H. banksii 39,377–387. Brand-Williams, W., Cuvelier, M. & Berset, C. (1995). Use of a free and Padina sp. had the higher antioxidant activities radical method to evaluate antioxidant activity. LWT-Food Science compared to the other species and comparable to the and Technology, 28,25–30. positive controls at all concentrations tested. Phenolic Chandini, S.K., Ganesan, P. & Bhaskar, N. (2008). In vitro antioxi- compounds mainly contributed for antioxidant activity dant activities of three selected brown seaweeds of India. Food 107 – of the extracts, while fucoxanthin was observed at all Chemistry, , 707 713. Chew, Y., Lim, Y., Omar, M. & Khoo, K. (2008). Antioxidant activ- six algae and showed the medium antioxidant capac- ity of three edible seaweeds from two areas in South East Asia. ity. Therefore, the extracts from S. vestitum, H. banksii LWT-Food Science and Technology, 41, 1067–1072. and Padina sp. will be utilised for isolation and purifi- Cho, M., Kang, I.J., Won, M.H., Lee, H.S. & You, S. (2010). The cation of bioactive components (focusing on phenolic antioxidant properties of ethanol extracts and their solvent-parti- tioned fractions from various green seaweeds. Journal of Medicinal compounds and fucoxanthin) which could be widely Food, 13, 1232–1239. applied in food and pharmaceutical fields. Cox, S., Abu-Ghannam, N. & Gupta, S. (2010). An assessment of the antioxidant and antimicrobial activity of six species of edible Irish seaweeds. Food Research International, 17, 205–220. Acknowledgments Dang, T.T., Vuong, Q.V., Schreider, M.J., Bowyer, M.C., Van Altena, I.A. & Scarlett, C.J. (2017). Optimisation of ultrasound- The authors would like to acknowledge the following assisted extraction conditions for phenolic content and antioxidant funding support: Ramaciotti Foundation (ES2012/ activities of the alga Hormosira banksii using response surface 0104). The authors also kindly thank University of methodology. Journal of Applied Phycology,1–13. https://doi.org/ Newcastle and the Vietnamese Government for award- 10.1007/s10811-017-1162-y. Dellai, A., Laajili, S., Le Morvan, V., Robert, J. & Bouraoui, A. ing a VIED-TUIT scholarship to Thanh Trung (2013). Antiproliferative activity and phenolics of the Mediter- DANG. ranean seaweed Laurencia obusta. Industrial Crops and Products, 47, 252–255. Farvin, K.S. & Jacobsen, C. (2013). Phenolic compounds and Conflict of interest antioxidant activities of selected species of seaweeds from Danish coast. Food Chemistry, 138, 1670–1681. The authors declare no conflict of interest. Fayaz, M., Namitha, K., Murthy, K.C. et al. (2005). Chemical com- position, iron bioavailability, and antioxidant activity of Kappaphy- cus alvarezzi (Doty). Journal of Agricultural and Food Chemistry, References 53, 792–797. Foo, S.C., Yusoff, F.M., Ismail, M. et al. (2015). Production of Agregan, R., Lorenzo, J.M., Munekata, P.E., Dominguez, R., Car- fucoxanthin-rich fraction (FxRF) from a diatom, Chaetoceros cal- ballo, J. & Franco, D. (2016). Assessment of the antioxidant citrans (Paulsen) Takano 1968. Algal Research, 12,26–32.

International Journal of Food Science and Technology 2018 © 2017 Institute of Food Science and Technology Comparison of antioxidant activity of algae T. T. Dang et al. 181

Foo, S.C., Yusoff, F.M., Ismail, M. et al. (2017). Antioxidant Murugan, K. & Iyer, V.V. (2013). Differential growth inhibition of capacities of fucoxanthin-producing algae as influenced by their cancer cell lines and antioxidant activity of extracts of red, brown, carotenoid and phenolic contents. Journal of Biotechnology, 241, and green marine algae. In Vitro Cellular & Developmental Biology- 175–183. Animal, 49, 324–334. Fung, A., Hamid, N. & Lu, J. (2013). Fucoxanthin content and Rajauria, G., Jaiswal, A.K., Abu-Gannam, N. & Gupta, S. (2013). antioxidant properties of Undaria pinnatifida. Food Chemistry, 136, Antimicrobial, antioxidant and free radical-scavenging capacity of 1055–1062. brown seaweed Himanthalia elongata from western coast of Ire- Gupta, S. & Abu-Ghannam, N. (2011). Bioactive potential and pos- land. Journal of Food Biochemistry, 37, 322–335. sible health effects of edible brown seaweeds. Trends in Food Rajauria, G., Foley, B. & Abu-Ghannam, N. (2016). Characteriza- Science & Technology, 22, 315–326. tion of dietary fucoxanthin from Himanthalia elongata brown sea- Huang, J., Liu, Y. & Wang, X. (2008). Selective adsorption of tan- weed. Food Research International, 99, 995–1001. nin from flavonoids by organically modified attapulgite clay. Jour- Seely, G., Duncan, M. & Vidaver, W. (1972). Preparative and ana- nal of Hazardous Materials, 160, 382–387. lytical extraction of pigments from brown algae with dimethyl sul- Jaswir, I., Noviendri, D., Salleh, H.M., Taher, M., Miyashita, K. & foxide. Marine Biology, 12, 184–188. Ramli, N. (2013). Analysis of fucoxanthin content and purification Sudhakar, M., Ananthalakshmi, J. & Nair, B. (2013). Extraction, of all-trans-fucoxanthin from Turbinaria turbinata and Sargassum purification and study on antioxidant properties of fucoxanthin plagyophyllum by SiO2 open column chromatography and reversed from brown seaweeds. Journal of Chemical and Pharmaceutical phase-HPLC. Journal of Liquid Chromatography & Related Tech- Research, 5, 169–175. nologies, 36, 1340–1354. Terasaki, M., Hirose, A., Narayan, B. et al. (2009). Evaluation of Jimenez-Escrig, A., Jimenez-Jimenez, I., Pulido, R. & Saura-Calixto, F. recoverable functional lipid components of several brown sea- (2001). Antioxidant activity of fresh and processed edible seaweeds. weeds (Phaeophyta) from Japan with special reference to fucoxan- Journal of the Science of Food and Agriculture, 81, 530–534. thin and fucosterol contents. Journal of Phycology, 45, 974–980. Kahk€ onen,€ M.P., Hopia, A.I., Vuorela, H.J. et al. (1999). Antioxi- Wang, T., Jonsdottir, R. & Olafsd ottir, G. (2009). Total phenolic dant activity of plant extracts containing phenolic compounds. compounds, radical scavenging and metal chelation of extracts Journal of Agricultural and Food Chemistry, 47, 3954–3962. from Icelandic seaweeds. Food Chemistry, 116, 240–248. Kim, S.M., Kang, S.W., Kwon, O.N., Chung, D. & Pan, C.H. (2012). Zhang, W.W., Duan, X.J., Huang, H.L., Zhang, Y. & Wang, B.G. Fucoxanthin as a major carotenoid in Isochrysis aff. galbana: charac- (2007). Evaluation of 28 marine algae from the Qingdao coast for terization of extraction for commercial application. Journal of the antioxidative capacity and determination of antioxidant efficiency Korean Society for Applied Biological Chemistry, 55, 477–483. and total phenolic content of fractions and subfractions derived Kumar, S.R., Hosokawa, M. & Miyashita, K. (2013). Fucoxanthin: from Symphyocladia latiuscula (Rhodomelaceae). Journal of a marine carotenoid exerting anti-cancer effects by affecting multi- Applied Phycology, 19,97–108. ple mechanisms. Marine Drugs, 11, 5130–5147. Zhishen, J., Mengcheng, T. & Jianming, W. (1999). The determina- Mohamed, S., Hashim, S.N. & Rahman, H.A. (2012). Seaweeds: a tion of Flavonoid contents in mulberry and their scavenging effects sustainable functional food for complementary and alternative on superoxide radicals. Food Chemistry, 64, 555–559. therapy. Trends in Food Science & Technology, 23,83–96. Munir, N., Sharif, N., Naz, S. & Manzoor, F. (2013). Algae: a potent antioxidant source. Sky Journal of Microbiology Research, 1,22–31.

© 2017 Institute of Food Science and Technology International Journal of Food Science and Technology 2018 Paper II:

“The Effects of Drying on Physico-Chemical Properties and Antioxidant Capacity of the Brown Alga (Hormosira banksii (Turner) Decaisne)”. Journal of Food Process and Preservation 41(4): e13025. doi.org/10.1111/jfpp.13025.

Journal of Food Processing and Preservation ISSN 1745-4549

THE EFFECTS OF DRYING ON PHYSICO-CHEMICAL PROPERTIES AND ANTIOXIDANT CAPACITY OF THE BROWN ALGA (HORMOSIRA BANKSII (TURNER) DECAISNE) THANH T. DANG1,2, QUAN VAN VUONG1, MARIA J. SCHREIDER1, MICHAEL C. BOWYER1, IAN A. VAN ALTENA1 and CHRISTOPHER J. SCARLETT1,3

1School of Environmental and Life Sciences, Faculty of Science and Information Technology, University of Newcastle, Ourimbah, NSW, Australia 2Department of Seafood Processing Technology, Faculty of Food Technology, NhaTrang University, Nha Trang, Khanh Hoa, Vietnam

3Corresponding author. ABSTRACT TEL: 1 61 2 4348 4680; FAX: 1 61 2 4348 4145; Hormosira banksii is a rich source of polyphenols, which can be utilized in the EMAIL: [email protected] food or pharmaceutical industries. This study aimed to determine the impact of six drying methods on properties of the alga H. banksii. Our data revealed that Received for Publication January 8, 2016 drying conditions significantly affected recovery yield, residual moisture, extrac- Accepted for Publication March 14, 2016 tion yield, total phenolic content (TPC) as well as antioxidant capacity of H. < doi:10.1111/jfpp.13025 banksii (P 0.05). Optimal conditions for oven, vacuum and de-humidification were 40, 50 and 50C, respectively, and microwave power is 840 W. Under optimal conditions, H. banksii prepared by freeze, de-humidification and vacuum had sig- nificantly higher levels of TPC, total flavonoid content (TFC) and proanthocyani- dins as well as possessing stronger antioxidant capacity in comparison with those prepared by sun, microwave and oven drying methods. As freeze drying is costly and time-consuming, de-humidification (50C, air in and out of 11.1 and 15.4%) and vacuum (50C, 10 psi) were recommended for drying H. banksii

PRACTICAL APPLICATIONS Algae possess various antioxidants with potential benefits for health. Drying is considered as a method for preserving materials, transport with low costs and especially first step for extraction, isolation and purification of active compounds. Thus, it is important to investigate the effects of drying conditions on the proper- ties of the dried alga H. banksii. From the findings, the different drying conditions significantly affected the phytochemical profile and antioxidant activity of the dried alga and the optimal drying conditions could be applied for preparation of dried H. banksii for further processing (extraction, fractionation and isolation of bioactive compounds) as well as potential industrial applications (as a reference for drying H. banksii and other algae).

INTRODUCTION et al. 2009; Ganesan et al. 2010; Sithranga Boopathy and Hormosira banksii (Fig. 1) is a brown fucoid alga (order: Kathiresan 2011; Abirami and Kowsalya 2012; Chung et al. Fucales; class: Phaeophyceae) widely distributed in the inter- 2012; Guerra Dore et al. 2013), diabetes, obesity, improved tidal areas along the southern coast of Australia (Millar and endothelial function, reduced blood pressure (Farasat et al. Kraft 1994). Secondary metabolites, especially phenolic 2014) and anti-HIV properties (Hassan Khan and Ather compounds, have been found abundantly within brown 2007). algae (Gupta and Abu-Ghannam 2011; Balboa et al. 2013). Drying is the first step in preparing natural materials for Phenolic compounds are known to possess antioxidant phytochemical extraction, isolation and identification of capacity and have been used in the treatment of some bioactive compounds. Optimization of drying conditions is chronic diseases, including various types of cancer (Sheih required to maximize preservation of the phytochemical

Journal of Food Processing and Preservation 2017; 41: e13025; VC 2016 Wiley Periodicals, Inc. 1of11 THE EFFECT OF DRYING ON PROPERTIES OF THE ALGA T.T. DANG ET AL.

washed thoroughly with fresh water, then stored at 220C until processed.

Procedures and Methods for Drying Samples To determine the impact of different drying methods on physico-chemical and antioxidant properties of H. banksii, six different drying methods were employed: sun-, micro- wave-, oven-, de-humidification-, vacuum- and freeze- drying. Prior to drying, frozen samples were thawed at room temperature overnight. The samples were dried to constant weight by the different methods. The weight of fresh and dried samples were recorded, and then stored at 220C for further analysis.

FIG. 1. HORMOSIRA BANKSII ALGA Sun Drying. Algal samples (25 g) were placed in a single layer on a clean aluminum tray and dried in direct sunlight profile and therefore represents an important area of study to constant weight (temperature 32 6 2C, approximately in natural product chemistry. Phenolic compounds are sen- 42 h). sitive to heat; thus, their retention can be significantly affected by drying conditions (Jimenez-Escrig et al. 2001). Freeze Drying. Algal samples (25 g) were immersed in Several drying methods have been employed for drying liquid nitrogen and then freeze dried for 48 h using a freeze algae. For example, freeze drying, oven drying and green- dryer (Thomas Australia Pty. Ltd., Seven Hills, NSW, Aus- 2 house drying were applied to brown algal species Sargassum tralia) set to a chamber pressure of 2 3 10 1 mbar and a muticum and Bifurcaria bifurcata (Le Lann et al. 2008); cryo-temperature of 250C. hydrothermal drying has been employed to dry Laminaria saccharina, Laminaria digitata and Himanthalia elongata Microwave Drying. The alga was shredded using a com- (Rajauria et al. 2010); sun drying, oven drying and freeze mercial blender (John Morris Scientific, Chatswood, NSW, drying have been used to dry Sargassum hemiphyllum Australia) prior to drying. Shredded alga sample (10 g) was (Turn.) C. Ag. (Chan et al. 1997); while de-humidified air then placed on the microwave plate in a single layer and drying was applied to dry red alga Eucheuma cottonii dried using a microwave oven (Sharp Caurousel inverter (Djaeni and Sari 2015). microwave 1200 W) set at powers ranging between 600 and There is a lack of information on drying alga H. banksii. 1,200 W. Samples were dried using a pulse irradiation proto- Therefore, the current study aimed to determine the impact col of 5 s on and 15 s off (to avoid sample burning), repeated of different drying methods on physical properties (recovery 36–48 times to constant weight (3–4 min, total irradiation yield %, residual moisture % and extraction yield %), chem- time). ical properties (phenolic compounds and other second metabolites) and antioxidant capacity of H. banksii. The Oven Drying. Algal samples (25 g) were placed in alumi- findings of this study could be used to indicate the most num trays in a single layer and then dried at different tem- appropriate drying methods for preparation of dried H. peratures 40C (drying time: 14 h); 50C (12 h); 60C (11 h); banksii for further processing. 70C (10 h) and 80C (9.5 h) using an oven drier (LABEC, Laboratory Equipment Pty Ltd., Marrickville, NSW, Australia) to constant weight. MATERIALS AND METHODS Vacuum Drying. Algal samples (25 g) were placed in alu- Algal Samples minum trays in a single layer and then dried at temperatures ranging between 40C (drying time: 12 h); 50C (11 h); 60C The fresh alga (H. banksii) was collected at Bateau Bay, Cen- (10 h); 70C (9.5 h) and 80C (9 h) at a pressure of 10 psi using tral Coast, New South Wales, Australia in March, 2014. After a vacuum oven (Thermoline, Australian Marketing Group, collection, the sample was washed with seawater to remove Wetherill Park, NSW, Australia) to a constant weight. natural residues (sand and epiphytes), then boxed to protect from light and immediately transported to the laboratory De-Humidification Drying. The humidity of the air, in for processing. At the laboratory, the alga was further and out, 11.1 and 15.4% respectively, was pre-set before

2of11 Journal of Food Processing and Preservation 2017; 41: e13025; VC 2016 Wiley Periodicals, Inc. T.T. DANG ET AL. THE EFFECT OF DRYING ON PROPERTIES OF THE ALGA drying. Algal samples (25 g) were dried at four different To determine antioxidant capacity, a working solution temperatures, 30C (drying time: 14 h); 40C (12 h); 50C was prepared by diluting the algal extraction 20-fold with (10 h) and 60C (9.5 h) to constant weight. ethanol (70%). Three antioxidant assays were applied to All drying methods at their optimal drying conditions determine the impact of different drying methods on anti- were then compared to identify the best drying methods to oxidant capacity of H. banksii. ABTS and DPPH assays were yield high levels of total phenolic content (TPC) from H. applied (Thaipong et al. 2006), as well as FRAP assay was banksii. conducted similarly to the method described by Gan and Latiff (2011). Methanol and trolox were used as the control Determination of Chemical and Antioxidant and standard, respectively, for each of the assay methods, Properties with antioxidant capacity for each expressed as trolox equiv- alent per gram of dried alga (mg TE/g dried alga). To determine the impact of individual drying methods on chemical profile of H. banksii, individual dried samples pre- pared from each different drying method were fine ground Determination of Physical Properties and sieved (diameter 600 mm) using a 600 mm EFL 2000 stainless steel mesh sieve (Endecotts Ltd., London, UK). The Hormosira banksii samples prepared using different drying alga powders were extracted under identical conditions and methods were tested to determine the impact of different the phytochemical profile of the extracts subsequently ana- drying methods on recovery yield, residual moisture and lyzed. Briefly, dried H. banksii samples (1 g) were extracted extraction yield. The analytical methods were performed as in 50 mL ethanol 70% in an ultrasonic bath (Soniclean, outlined previously (Vuong et al. 2013) with some modifi- 220 V, 50 Hz and 250W, Soniclean Pty Ltd., Australia) set at cations. Briefly, recovery yield was expressed as a percentage temperature of 50C and power of 250 W for 90 min. The of dried alga mass over the weight of fresh alga mass shown extracts were then filtered twice using filter paper (Lomb in Eq. (1): Scientific, Taren Point, NSW, Australia) to remove sus- 5 ð = Þ 3 ; pended solids prior to analysis. DY % DS FS 100% (1) For testing TPC, total flavonoid content (TFC) and where DY% (recovery yield); DS (g) is weight of dried sam- proanthocyanidins, working solutions were prepared by ple after drying and FS (g) is weight of fresh sample before diluting 10-fold the extracts with ethanol (70%). TPC was drying. determined using Folin–Ciocalteu method as described in The moisture of fresh samples and residual moisture of previous studies (Thaipong et al. 2006; Vuong et al. 2013). dried samples were tested according to AOAC official Gallic acid was used as the standard, with TPC expressed as method No. 934.06 (AOAC 1990) using the hot air drying mg of gallic acid equivalents (mg GAE). TFC and proantho- cyanidins were determined as described in previous studies method at 70C for 72 h. Finally, extraction yield was calcu- (Thaipong et al. 2006; Vuong et al. 2013). Catechin was used lated by drying 5 mL of the filtered extract at 80C under as the standard, with the levels of TFC and proanthocyani- reduced pressure (5 psi, 24 h) using a vacuum oven, with the dins expressed as mg of Catechin equivalent (mg CAE) per value expressed as a percentage of the original sample mass gram of the dried alga. shown in Eq. (2). Antioxidant capacity was determined using three antioxi- EY % 5 ðDE=DSÞ 3100%; (2) dant assays, namely ABTS (2,20-azino-bis(3-ethylbenzothia- zoline-6-sulphonic acid), DPPH (2,2-diphenyl-1- where EY% (extraction yield, %); DE (g) is weigh of dried picrylhydrazyl) and FRAP (ferric reducing antioxidant extract and DS (g) is weigh of dried sample before power). More than one antioxidant assay was conducted to extraction. increase the confidence of resulting data, because of the limitation of each method in capturing all potential antioxi- dant species present (Pisoschi 2011). For example, the ABTS Statistical Analysis assay has applicability over a wide pH range and is tolerant to different types of solvents. In the case of DPPH, many All experiments were performed at least in triplicate. The antioxidants react quickly with peroxyl radicals in solution data were expressed as mean 6 standard deviation (n 5 3). and may react slowly or be inert to DPPH due to steric inac- A one-way ANOVA and LSD post hoc test were employed cessibility (Prior et al. 2005). The FRAP assay by contrast (SPSS Statistical Software, Version 16) to analyze the differ- measures the reducing capability based upon the ferric ion ences between drying methods. Differences between the and is not relevant to mechanistic antioxidant activity under mean levels of the components in different experiments normal physiological conditions (Prior et al. 2005). were taken to be statistically significant at P < 0.05.

Journal of Food Processing and Preservation 2017; 41: e13025; VC 2016 Wiley Periodicals, Inc. 3of11 THE EFFECT OF DRYING ON PROPERTIES OF THE ALGA T.T. DANG ET AL.

TABLE 1. THE EFFECTS OF TEMPERATURE ON TOTAL PHENOLIC COMPOUNDS (TPC) AND ANTIOXIDANT PROPERTIES OF THE ALGA H. BANKSII FROM OVEN DRYING Antioxidant activity

TPC (mg GAE/g ABTS (mg TE/g DPPH (mg TE/g FRAP (mg TE/g Temperature (C) dried sample) dried sample) dried sample) dried sample) 40 10.00 6 0.30a 35.40 6 1.78a 23.75 6 0.60a 5.45 6 0.26a 50 9.51 6 0.29a 30.39 6 0.31b 22.57 6 0.63ab 5.57 6 0.63a 60 8.92 6 0.16b 26.58 6 0.75cd 21.63 6 0.76b 4.53 6 0.16b 70 8.01 6 0.23c 27.89 6 0.74c 19.13 6 0.45c 3.23 6 0.35c 80 8.14 6 0.14c 25.76 6 1.11d 16.45 6 0.30d 3.45 6 0.30c

Note: The data are means 6 standard deviations (n 5 3). The data in the same column not sharing the same superscript letter are significantly different at P < 0.05.

RESULTS AND DISCUSSION auto-oxidation kinetics at elevated temperatures (Shahidi et al. 1992). On the other hand, advantages of oven drying is Impact of Oven Drying Conditions on TPC that it is faster and more suitable for protein extraction in and Antioxidant Properties algae due to the cell wall being destroyed more intensively than that of freeze drying (Wong and Cheung 2001). The Drying the alga with the temperature ranging from 40 to 80C optimal oven drying temperature was determined to be 40C was applied. The results showed that oven temperature signif- for H. banksii, with these conditions subsequently used in icantly affected TPC extracted from H. banksii (Table 1; comparison with other drying methods. P < 0.05). Levels of TPC were not significantly different in the temperature range 40–50C (P > 0.05), however the con- tent of TPC significantly decreased at temperatures between Impact of Vacuum Drying Conditions on TPC 60 and 80C. Similarly, antioxidant capacity of H. banksii was and Antioxidant Properties found to be lower when drying temperatures exceeded 50C. These findings were supported by the previous studies of Lim Table 2 indicates that TPC and antioxidant capacity of H. and Murtijaya (2007) and Wong and Cheung (2001) who banksii were significantly affected by drying temperature, found that drying temperature in an oven above 40C resulted with the levels of both parameters slightly increasing across in a rapid degradation in levels of TPC and lower antioxidant the temperature range 40–50C. TPC and antioxidant capacity. Drying with high temperature, more phenolic com- capacity of H. banksii were significantly reduced when the pounds may combine with protein or be oxidized to qui- drying temperature exceeded 60C. The application of vac- nones reacted with active groups of protein molecules that uum drying, an oxygen-free environment and low tempera- caused the reduction of total phenolic compounds (Wong ture drying could prevent the oxidation and degradation of and Cheung 2001). However, the phenolic compounds in red antioxidants (Hossain et al. 2010) and improve the TPC lev- grape pomace peels reported by Larrauri et al.(1997)and els and antioxidant capacity of algal extracts. Optimal vac- mulberry leaf by Katsube et al. (2009) were not significantly uum drying conditions were determined to be 50C at affected when dried at 60C. It could be that the thermal sensi- pressure of 10 psi. These conditions were used for compari- tivity of phenolics in H. banksii, which underwent accelerated son with other drying methods.

TABLE 2. THE EFFECTS OF TEMPERATURE ON TOTAL PHENOLIC COMPOUNDS (TPC) AND ANTIOXIDANT PROPERTIES OF THE ALGA H. BANKSII FROM VACUUM DRYING Antioxidant activity

TPC (mg GAE/g ABTS (mg TE/g DPPH (mg TE/g FRAP (mg TE/g Temperature (C) dried sample) dried sample) dried sample) dried sample) 40 9.02 6 1.07a 39.29 6 2.02a 25.23 6 1.19a 3.93 6 0.16a 50 12.06 6 0.29b 47.08 6 0.93b 29.70 6 0.34b 5.40 6 0.38b 60 11.29 6 1.46bc 46.69 6 3.13b 22.51 6 1.65ac 4.46 6 0.96ab 70 11.21 6 0.59bc 40.43 6 1.64a 21.16 6 0.93c 3.61 6 0.24a 80 10.69 6 0.89ac 37.58 6 1.41a 21.81 6 1.24c 3.97 6 0.62a

Note: The data are means 6 standard deviations (n 5 3). The data in the same column not sharing the same superscript letter are significantly different at P < 0.05.

4of11 Journal of Food Processing and Preservation 2017; 41: e13025; VC 2016 Wiley Periodicals, Inc. T.T. DANG ET AL. THE EFFECT OF DRYING ON PROPERTIES OF THE ALGA

TABLE 3. THE EFFECTS OF TEMPERATURE ON TOTAL PHENOLIC COMPOUNDS (TPC) AND ANTIOXIDANT PROPERTIES OF THE ALGA H. BANKSII IN DE-HUMIDIFICATION DRYING Antioxidant activity

TPC (mg GAE/g ABTS (mg TE/g DPPH (mg TE/g FRAP (mg TE/g Temperature (C) dried sample) dried sample) dried sample) dried sample) 30 12.55 6 0.81a 38.24 6 1.64a 31.00 6 1.15a 4.68 6 0.37a 40 12.80 6 0.67a 40.97 6 1.07a 32.68 6 0.70a 4.82 6 0.45a 50 14.10 6 0.19b 49.34 6 0.75c 36.0 6 0.85b 6.28 6 0.08b 60 13.81 6 0.14b 47.34 6 0.32d 35.20 6 0.42b 6.03 6 0.41b

Note: The data are means 6 standard deviations (n 5 3). The data in the same column not sharing the same superscript letter are significantly different at P < 0.05.

Impact of De-Humidification Drying comparison of TPC and antioxidant activity against other Conditions on TPC and Antioxidant drying methods. Properties

Table 3 shows the levels of TPC and antioxidant capacity of Impact of Microwave Drying Conditions on algae H. banksii under de-humidification drying (11.1% TPC and Antioxidant Properties RH in and 15.4% out), with the temperature ranging from 30 to 60C. TPC and antioxidant capacity were found to be Microwave powers ranging between 600 and 1,200 W were similar in temperature range 30–40C indicating little ther- investigated for drying H. banksii, with the results presented mally promoted auto-oxidation. Maximum TPC and anti- in Table 4. They revealed that microwave powers signifi- oxidant activity was observed at a drying temperature of cantly affected TPC and antioxidant capacity. TPC remained 50C and there was no significant difference in TPC and unchanged in the power range 600–700 W was significantly antioxidant activity (DPPH and FRAP) between the tem- higher in the power range 840–1,080 W and was again lower peratures of 50 and 60C (P > 0.05). These findings could at 1,200 W. One advantage of using microwave for drying be explained that under low relative humidity and high was a short time of the procedure (a few minutes). In com- temperature (50 and 60C), H. banksii could be dried in a parison with other methods, TPC and antioxidant capacity shorter time period; while at the lower temperature, a lon- of microwave dried extracts reduced dramatically. The ger time required for drying caused a stronger degradation results were consistent to those reported by Chan et al. of antioxidants. The result was supported by Djaeni and (2009) who indicated that microwave drying procedure was Sari (2015) who reported that with lower humidity, the just 2 min for removing moisture content, but degrading all drying time was shortened and de-humidified air was suit- thermo-sensitive antioxidants in the leaves and tea of ginger able for drying algae at temperatures lower than 70C with species. On the other hand, TPC values of microwave dried little effect on nutritional composition of the algae. Fud- samples were not significantly affected by microwave powers holi et al. (2011) also indicated that the best condition for and were higher than those by oven for drying onion slices drying algae was temperature 60C and the relative humid- (Arslan and Ozcan€ 2010). From the findings, optimal ity of 10%. The optimal temperature of 50C was chosen for microwave power determined to be 840 W.

TABLE 4. THE EFFECTS OF TEMPERATURE ON TOTAL PHENOLIC COMPOUNDS (TPC) AND ANTIOXIDANT PROPERTIES OF THE ALGA H. BANKSII IN MICROWAVE DRYING Antioxidant activity

Microwave TPC (mg GAE/g ABTS (mg TE/g DPPH (mg TE/g FRAP (mg TE/g power (W) dried sample) dried sample) dried sample) dried sample) 600 2.65 6 0.17a 13.90 6 0.41d 5.04 6 0.39c 1.66 6 0.38a 720 2.64 6 0.10a 14.72 6 1.50abd 5.80 6 0.69ac 2.59 6 0.10b 840 3.99 6 0.01b 18.45 6 1.06c 7.94 6 0.28b 2.46 6 0.04b 960 3.72 6 0.39b 18.68 6 0.58c 6.32 6 0.47a 2.54 6 0.31b 1080 3.72 6 0.26b 15.65 6 0.93b 5.52 6 0.46ac 1.25 6 0.18a 1200 2.89 6 0.18a 12.21 6 1.24a 5.18 6 0.96ac 1.17 6 0.15a

Note: The data are means 6 standard deviations (n 5 3). The data in the same column not sharing the same superscript letter are significantly different at P < 0.05.

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TABLE 5. EFFECT OF DRYING METHODS ON TOTAL PHENOLIC COMPOUNDS (TPC), TOTAL FLAVONOID CONTENT (TFC) AND TOTAL PROANTHOCYANIDIN CONTENT OF H. BANKSII TPC (mg GAE/g TFC (mg CAE/g Proanthocyanidins Method dried sample) dried sample) (mg CAE/g dried sample) Sun drying 1.17 6 0.08a 0.76 6 0.05a 1.58 6 0.04a Microwave (840 W) 3.99 6 0.01b 3.51 6 0.15b 4.22 6 0.15b Oven (40C) 10.00 6 0.30c 2.61 6 0.07c 5.84 6 0.10c Vacuum (50C) 12.06 6 0.29d 8.88 6 0.19d 9.08 6 0.21d Dehumidification (50C) 14.10 6 0.19e 5.96 6 0.08e 12.29 6 0.06e Freeze drying 21.11 6 0.17f 5.18 6 0.08f 17.25 6 0.57f

Note: The data are means 6 standard deviations (n 5 3). The data in the same column not sharing the same superscript letter are significantly different at P < 0.05.

Comparison of Different Drying Methods on samples were subjected to multiple wavelengths and ener- Chemical Properties gies across the electromagnetic spectrum (UV to IR) that are likely to accelerate the degradation of components present Phenolic compounds in algae have been closely linked to in seaweed (Chan et al. 1997). In addition, the enzymes antioxidant capacity, which is in turn associated with poten- (such as polyphenol oxidases) in the fresh materials were tially beneficial health properties (Lijun et al. 2005; Kim not immediately deactivated in sun drying that caused the et al. 2011; Jung et al. 2013). Thus, it is necessary to identify degradation of phenolic compounds (Lim and Murtijaya drying conditions that have the least effect on TPC loss dur- 2007; Hossain et al. 2010). ing the drying process. The impact of different drying methods on TFC (Table Table 5 highlighted the effect of the different optimized 5) revealed that the different optimal drying methods sig- drying conditions on TPC of H. banksii. The present data nificantly affected the retention of TFC. Between the six showed that H. banksii extract possessed high phenolic con- drying methods, vacuum dried algae retained the highest tent in comparison to that of other algae and plants (Lim concentrationofTFC(8.88mgCAE/gdriedalga).Theval- and Murtijaya 2007; Gupta and Abu-Ghannam 2011). ues for de-humidification and freeze drying were 5.96 and Freeze drying produced the highest TPC (21 mg GAE/g of 5.18 mg CAE/g, respectively. Microwave-, oven- and sun dried alga), followed by de-humidification (14.10 mg GAE/ drying had low TFC levels (3.51, 2.61 and 0.76 mg CAE/g g); vacuum drying (12.06 mg GAE/g) and oven drying dry material, respectively). Thermal and enzymatic factors (10.00 mg GAE/g). Microwave and sun drying showed the have also been reported to accelerate flavonoid degrada- lowest levels of TPC (3.99 and 1.17 mg GAE/g, respectively). tion (Mohd Zainol et al. 2009). Gupta et al. (2011) stated The different irradiative processes associated with the that the TFC in the fresh Himanthalia elongata was respective drying methods can explain variations in reten- 0.49 g 6 0.019 QE/100 g dry algae and a reduction in the tion of TPC. Freeze drying and vacuum drying were con- TFC of 49 and 30% was seen at 25 and 40C, respectively, ducted at low temperatures with limited exposure to other when drying by oven. It can be explained that the low dry- parts of the electromagnetic spectrum (e.g., visible and UV ing temperatures could not completely inactivate the oxi- light) and reduced exposure to atmospheric oxygen, both of dative enzymes that caused oxidation of the phenolic which are associated with phenol degradation (Lim and substances and led to a low phenolic content. In addition, Murtijaya 2007; Le Lann et al. 2008). Freeze drying is con- the combination of polyphenols with other compounds sidered the best drying method for algae because it is effi- (proteins) or the alterations in the chemical structure of cient in preserving thermo-sensitive compounds (Franks polyphenols, which cannot be extracted or determined by 1998; Bennamoun et al. 2015). Drying seaweed using de- available methods, may also have led to lower phenolic humidified air had a positive result as the low humidity led content (Gupta et al. 2011). Cox et al. (2012) also reported to the shorter drying time, which again limited potential that fresh Himanthalia elongata contained 42.29 mg QE/g degradation (Fudholi et al. 2011; Djaeni and Sari 2015). In of extract. Air drying for 12 and 24h significantly reduced contrast, microwave drying was conducted using irradia- the TFC in the range of 2.45–9.35%, respectively, in com- tion, which might lead to thermal degradation of active parison with the fresh sample. The time and power of the compounds. A previous study on Phyllanthus amarus by microwave had the strongest effect on both TPC and TFC Lim and Murtijaya (2007) indicated that microwave drying of the loquat flower tea (Eriobotrya japonica (Lindl.) caused the greatest decrease in TPC and antioxidant activity, Thunb.). The degradation of some polyphenols and flavo- which was exhibited by the reduction in both free radical noids was increased with the high power or low power scavenging activity and FRAP. In the case of sun drying, with longer time (Zheng et al. 2015).

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TABLE 6. EFFECT OF DRYING METHODS ON ABTS ANTIOXIDANT CAPACITY, DPPH SCAVENGING CAPACITY AND FERRIC ION REDUCING ANTIOXIDANT POWER (FRAP) OF H. BANKSII Antioxidant activity

ABTS (mg TE/g DPPH (mg TE/g FRAP (mg TE/g Method dried sample) dried sample) dried sample) Sun drying 8.58 6 0.21a 1.35 6 0.15a 3.26 6 0.18a Microwave (840 W) 18.45 6 1.06b 7.94 6 0.28b 2.46 6 0.04b Oven (40C) 35.40 6 1.78c 23.75 6 0.60c 5.45 6 0.26c Vacuum (50C) 47.08 6 0.93d 29.70 6 0.34d 5.40 6 0.38c Dehumidification (50C) 49.34 6 0.75d 36.00 6 0.85e 6.28 6 0.08d Freeze drying 72.76 6 0.50e 49.92 6 1.20f 10.58 6 0.19e

Note: The data are means 6 standard deviations (n 5 3). The data in the same column not sharing the same superscript letter are significantly different at P < 0.05.

Proanthocyanidin content (Table 5) followed a similar pounds as antioxidants was emphasized in algae, with the trend to the TPC findings. The highest levels were identified highest TAC in freeze-dried extracts (Jimenez-Escrig et al. in algal samples prepared by freeze drying (17.25 mg CAE/ 2001; Kuda et al. 2007; Le Lann et al. 2008). De- g). The high proanthocyanidin values were produced by de- humidification and vacuum dried algae also produced the humidification (12.29 mg CAE/g) and vacuum drying high antioxidant activities due to the limitation of oxygen, (9.08 mg CAE/g dried alga). Sun drying again produced the short drying time and proper temperature. These TAC val- lowest concentration (1.58 mg CAE/g), while microwave ues displayed a similar trend in comparison with that of dry- and oven drying recorded intermediate values (4.22 and ing un-matured citrus fruits using freeze-, hot air and sun 5.84 mg CAE/g, respectively). Rajauria et al. (2010) reported drying (Sun et al. 2015). However, with loquat flower tea that proanthocyanidins (total condensed tannin content) in both TAC and DPPH of microwave dried samples were raw H. elongata, L. saccharina and L. digitata algae was higher than those of vacuum and hot air dried samples 10.5 6 0.73, 11.5 6 0.77 and 8.1 6 0.73 mg CAE/g, respec- (Zheng et al. 2015). The reason may be that there were more tively. Values of proanthocyanidins in these algae increased thermo-sensitive antioxidants in H. banksii than those of (1.9–2.6 fold) when the algae was treated at 95C (15 min). loquat flower tea. Linear regression analysis of antioxidant From H. elongata,Coxet al. (2012) showed that the fresh activity with the phytochemical content of extracts pro- extract contained 55.7 mg CAE/g. This value was reduced duced r values that showed statistically significant correla- 7.73 and 8.65% in samples dried for 12 and 24 h, respec- tions (Jimenez-Escrig et al. 2001). The relationships tively. Using microwave at 450 and 900 W caused 20.27 and between the TPC, TFC and proanthocyanidins of H. banksii 22.54% reduction in proanthocyanidins, respectively. with its TAC were illustrated by correlation coefficients r2 5 0.992, 0.490 and 0.873, respectively (Table 7). TPC and proanthocyanidins had a very strong correlation to TAC, Comparison of Different Drying Methods on while TFC had a moderate one to its TAC, revealing that Antioxidant Properties TPC and proanthocyanidins are major contributors to TAC The results from the ABTS assay highlighted the impact of of H. banksii. different drying methods on total antioxidant capacity (TAC) of H. banksii (Table 6). These data showed that freeze dried samples exhibited the highest TAC (72.76 mg TE/g TABLE 7. CORRELATION COEFFICIENTS BETWEEN ANTIOXIDANT ACTIVITIES AND TOTAL PHENOLIC CONTENT (TPC), TOTAL FLAVONOID dried alga). TAC values of samples obtained by de- CONTENT (TFC) AND PROANTHOCYANIDINS OF H. BANKSII BY SIX humidification (49.34 mg TE/g), vacuum (47.08 mg TE/g) DRYING METHODS and oven drying (35.40 mg TE/g) were also high. Sun dried Antioxidant activity alga had the lowest TAC (8.58 mg TE/g). Samples using sun- , microwave-, oven-, vacuum- and de-humidification- FRAP (ferric drying had approximately 32, 35, 51, 75 and 88% TAC of ABTS DPPH reducing freeze dried alga, respectively, showing that different drying (antioxidant (scavenging antioxidant capacity) capacity) power) conditions significantly affected TAC (P < 0.05). It was clear that thermo-sensitive antioxidants could be preserved dur- TPC 0.992 0.993 0.920 TFC 0.490 0.473 0.205 ing freeze drying with perfect conditions: low temperature Proanthocyanidins 0.873 0.855 0.758 and the limitation of oxygen. The role of phenolic com-

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TABLE 8. IMPACT OF DRYING METHODS ON PHYSICAL PROPERTIES OF H. BANKSII Drying methods

Microwave Dehumidification Freeze (250C, Sun (840 W) Oven (40C) Vacuum (50C) (50C) 2.1021 mbar) Yield (%) 25.81 6 0.95a 21.06 6 0.78b 20.32 6 0.37b 19.41 6 0.23c 20.35 6 0.38b 19.46 6 0.43c Residual moisture (%) 16.16 6 0.36a 9.82 6 0.14b 7.68 6 0.03c 6.73 6 0.21d 7.57 6 0.32c 5.69 6 0.12e Extraction yield (%) 18.08 6 0.38a 23.87 6 1.24b 20.00 6 0.25c 24.38 6 0.18b 25.25 6 0.71b 29.00 6 0.71d

Note: The data are means 6 standard deviations (n 5 3). The data in the same column not sharing the same superscript letter are significantly different at P < 0.05.

The drying conditions significantly affected DPPH scav- wave dried ones. In addition, freeze-, de-humidification and enging activity of dried alga H. banksii (Table 6). Freeze- vacuum-drying were still the appropriate choice for drying dried algae had the highest DPPH radical scavenging algae as well as other plants (Chan et al. 1997; Le Lann et al. capacity (49.92 mg TE/g dried alga), while de- 2008; Hossain et al. 2010; Wu et al. 2014; Djaeni and Sari humidification and vacuum dried samples were 36.00 and 2015). 29.70 mg TE/g dried alga, respectively. Oven and microwave It was clear that the application of different drying condi- dried algae showed intermediate values (23.75 and 7.94 mg tions for the alga H. banksii significantly affected bioactive TE/g, respectively). Sun dried algae had the lowest DPPH compounds yield, especially unstable and thermo-sensitive radical scavenging activity (with only 1.35 mg TE/g). This antioxidants that led to a various levels of antioxidant trend was similar to that of TAC values of algal extracts. capacity in dried algae. There was a trend that the reduction TPC and proanthocyanidins of H. banksii were found to of TPC in alga H. banksii by various treatments was linked have a very high correlation with DPPH radical scavenging to the respective decrease in antioxidant activity (ABTS, activity (r2 5 0.993 and 0.855, respectively) while TFC had a DPPH and FRAP) of extracts. low correlation with DPPH radical scavenging activity (r2 5 0.473; Table 7). The findings were agreement with Effect of Drying Conditions on Physical Jimenez-Escrig et al. (2001) who stated that there was a Properties strong correlation between phenolic hydroxyl groups and free radical scavenge activity in algal extracts, while other The physical properties such as recovery yield, residual antioxidants such as ascorbic acid, carotenoids play a minor moisture and extraction yield of a dried sample are impor- role in antioxidant capacity of algae. The studies in Sargas- tant because these properties are directly linked to the post- sum muticum, Bifurcaria bifurcata, Laminaria sp. and Unda- harvest shelf life and the cost-effectiveness of the dried ria pinnatifida also reported that there was a close products. Therefore, the impact of different drying methods correlation between TPC and DPPH radical scavenging on these properties of H. banksii was assessed (Table 8). activity of algae (Le Lann et al. 2008; Amorim et al. 2012). Overall, the moisture of fresh sample was determined to be The results from the FRAP assay (Table 6) revealed that 81.43% and the recovery yield ranged between 20 and 26 kg the values of FRAP in freeze dried algae was the highest of dried algae from 100 kg of fresh algae. The recovery yield (10.58 mg TE/g dry material), followed by de- was significantly affected by drying conditions. Sun drying humidification, vacuum, oven and sun drying algae (6.28, produced the highest recovery yield (25.81%), followed by 5.40, 5.45 and 3.26 mg TE/g dry material, respectively). microwave drying (21.06%), de-humidification (20.35%), Microwave dried algae had the lowest value with 2.46 mg oven (20.32%) and freeze drying (19.46%), while vacuum TE/g dry material. There was no significant difference drying had the lowest recovery yield (19.41%). The levels of between vacuum and oven drying (P > 0.05). These findings the residual moisture remaining in the algal samples after indicated that FRAP of H. banksii were significantly affected drying can explain the differences in the recovery yield. by different drying conditions. Similarly to TAC and DPPH Upon drying to constant mass, sun dried algae had the high- radical scavenging activity, TPC and proanthocyanidins of est residual moisture (16.16%). These values for microwave, H. banksii had a high correlation with FRAP (r2 5 0.92 and oven, de-humidification and vacuum ranged between 6.73 0.758, respectively), revealing that TPC and proanthocyani- and 9.82%, while the lowest one for freeze dried algal sample dins are the major contributors to FRAP of H. banksii (Table was 5.69%. The difference in drying conditions also affected 7). The results were consistent with that observed for Phyl- texture as well as surface tension of the material that caused lanthus amarus by Lim and Murtijaya (2007) that the FRAP the change in recovery yield and residual moisture (Tello- value in sun dried samples was higher than that in micro- Ireland et al. 2011).

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Residual moisture significantly affects the stability of bio- de-humidification drying were 40, 50 and 50C, respectively, active compounds within dried materials during storage whereas optimal microwave drying conditions were 840 W. because it is positively linked to oxidation, enzyme and Under optimal drying conditions, H. banksii prepared by microbial activities that can degrade bioactive compounds freeze-, de-humidification and vacuum drying methods had (Toontom et al. 2012). Therefore, it is suggested that the significantly higher levels of TPC, TFC and proanthocyani- lower the residual moisture remaining in the dried samples, dins as well as possessed stronger antioxidant capacity in the less degradation of bioactive compounds could occur comparison with those prepared by sun, microwave and during transportation and/or storage. There is no previous oven drying methods. TPC mainly contributed for antioxi- study conducted on the link between residual moisture and dant activity of algal extracts. As freeze drying is costly and the stability of bioactive compounds of algae. Therefore, fur- time consuming, de-humidification (50C, air in and out of ther studies are recommended to identify the maximum 11.1 and 15.4%) and vacuum drying (50C, 10 psi) were rec- permissible level of residual moisture to minimize the deg- ommended for drying H. banksii for further extraction and radation of bioactive compounds within algae. isolation of phenolic compounds. It should be noted that Drying conditions might alter the surface matrix and tex- the current study determined the impact of drying condi- ture of dried materials and thus can affect the extraction tions on total phenolic compounds and other secondary efficiency when immersing the samples into the solvents. It metabolites and did not test the impact of drying conditions can be seen that extraction yield obtained from H. banksii on retention of individual phenolic compounds; therefore, was significantly influenced by different drying conditions further study is recommended. (Table 8). Under an identical extraction protocol, signifi- cantly higher extraction yields were obtained from algal samples prepared by freeze drying, de-humidification, vac- ACKNOWLEDGMENTS uum drying and microwave drying (29.00, 25.25, 24.38 and 23.87%, respectively) compared with those from sun or The authors acknowledge the following funding support: oven drying (18.08 and 20.00%, respectively). It was clear Ramaciotti Foundation (ES2012/0104) and the University that the methods with low temperature and short time of Newcastle. We also would like to thank Shane Murchie, gained higher extraction yield for H. banksii due to the University of Newcastle, for collecting the algal samples. We effects of the surface, texture as well as components of dried sincerely thank the Vietnamese Government through the samples. This was supported by Li et al. (2006) who indi- Vietnam International Education Development – Ministry cated that high temperature or long time made plant cell of Education and Training and Newcastle University for compounds adhere together with the absence of water, and awarding a VIED-TUITscholarship to Thanh Trung DANG. possibly making the extraction with solvent more difficult; as a result, the overall recoveries might be lower than drying REFERENCES conditions with low temperature or short time. However, Wong and Cheung (2001) with alga Sargassum hemiphyllum ABIRAMI, R. and KOWSALYA, A. 2012. Anticancer activity of stated that protein extraction yield in oven dried samples methanolic and aqueous extract of Ulva fasciata in albino was higher than that in freeze dried ones due to the cell wall mice. Int. J. Pharm. Pharm. Sci. 4(2), 681. of alga being more intensively damaged by high temperature AMORIM, K., LAGE-YUSTY, M.-A. and LOPEZ-HERN ANDEZ, J. 2012. Changes in bioactive in the oven in comparison to freeze treatments. This led to compounds content and antioxidant activity of seaweed after higher extraction yield from oven dried samples. The ingre- cooking processing. Cyta-J. Food, 10(4), 321–324. dient of components and texture in different materials may AOAC. 1990. Official Method of Analysis, 15th ed., Association of be the reason for the difference in extraction yield. In gen- Official Analytical Chemists, Washington, D. C. eral, all tested physical properties of H. banksii samples were ARSLAN, D. and OZCAN,€ M.M. 2010. Study the effect of sun, significantly affected by different drying methods and these oven and microwave drying on quality of onion slices. observations were used as one of the indicators for the LWT – Food Sci. Technol. 43(7), 1121–1127. determination of the most suitable drying methods for H. BALBOA, E.M., CONDE, E., MOURE, A., FALQUE, E. and banksii. DOMINGUEZ, H. 2013. In vitro antioxidant properties of crude extracts and compounds from brown algae. Food CONCLUSION Chem. 138(2), 1764–1785. BENNAMOUN, L., AFZAL, M.T. and LEONARD, A. 2015. Different drying conditions significantly affected the physi- Drying of alga as a source of bioenergy feedstock and food cal, chemical and antioxidant properties of the dried alga, supplement – A review. Renewable Sustainable Energy Rev. H. banksii. The optimal conditions for oven, vacuum and 50, 1203–1212.

Journal of Food Processing and Preservation 2017; 41: e13025; VC 2016 Wiley Periodicals, Inc. 9of11 THE EFFECT OF DRYING ON PROPERTIES OF THE ALGA T.T. DANG ET AL.

CHAN, E., LIM, Y.Y., WONG, S.K., LIM, K., TAN, S., LIANTO, HOSSAIN, M., BARRY-RYAN, C., MARTIN-DIANA, A.B. and F. and YONG, M. 2009. Effects of different drying methods on BRUNTON, N. 2010. Effect of drying method on the the antioxidant properties of leaves and tea of ginger species. antioxidant capacity of six Lamiaceae herbs. Food Chem. Food Chem. 113(1), 166–172. 123(1), 85–91. CHAN, J.C.-C., CHEUNG, P.C.-K. and ANG, P.O. 1997. JIMENEZ-ESCRIG, A., JIMENEZ-JIM ENEZ, I., PULIDO, R. Comparative studies on the effect of three drying methods on and SAURA-CALIXTO, F. 2001. Antioxidant activity of fresh the nutritional composition of seaweed Sargassum hemiphyllum and processed edible seaweeds. J. Sci. Food Agric. 81(5), (Turn.) C. Ag. J. Agric. Food Chem. 45(8), 3056–3059. 530–534. CHUNG, J.-G., PENG, H.-Y., CHU, Y.-C., HSIEH, Y.-M., JUNG, H.A., JIN, S.E., AHN, B.R., LEE, C.M. and CHOI, J.S. WANG, S.-D. and CHOU, S.-T. 2012. Anti-invasion and 2013. Anti-inflammatory activity of edible brown alga Eisenia apoptosis induction of chlorella (Chlorella sorokiniana)in bicyclis and its constituents fucosterol and phlorotannins in Hep G2 human hepatocellular carcinoma cells. J. Funct. LPS-stimulated RAW264. 7 macrophages. Food Chem. Toxicol. Foods 4(1), 302–310. 59, 199–206. COX, S., ABU-GHANNAM, N. and GUPTA, S. 2012. Effect of KATSUBE, T., TSURUNAGA, Y., SUGIYAMA, M., FURUNO, T. processing conditions on phytochemical constituents of edible and YAMASAKI, Y. 2009. Effect of air-drying temperature on Irish seaweed Himanthalia elongata. J. Food Process. Preserv. antioxidant capacity and stability of polyphenolic compounds 36(4), 348–363. in mulberry (Morus alba L.) leaves. Food Chem. 113(4), DJAENI, M. and SARI, D.A. 2015. Low temperature seaweed 964–969. drying using dehumidified air. Proc. Environ. Sci. 23, 2–10. KIM, S.M., SHANG, Y.F. and UM, B.H. 2011. A preparative FARASAT, M., KHAVARI-NEJAD, R.-A., NABAVI, S.M.B. and method for isolation of fucoxanthin from Eisenia bicyclis by NAMJOOYAN, F. 2014. Antioxidant activity, total phenolics and centrifugal partition chromatography. Phytochem. Anal. flavonoid contents of some edible green seaweeds from northern 22(4), 322–329. coasts of the Persian Gulf. Iran. J. Pharm. Res. 13(1), 163. KUDA, T., KUNII, T., GOTO, H., SUZUKI, T. and YANO, T. FRANKS, F. 1998. Freeze-drying of bioproducts: Putting principles 2007. Varieties of antioxidant and antibacterial properties of intopractice.Eur.J.Pharm.Biopharm.45(3), 221–229. Ecklonia stolonifera and Ecklonia kurome products harvested FUDHOLI, A., OTHMAN, M.Y., RUSLAN, M.H., YAHYA, M., and processed in the Noto peninsula, Japan. Food Chem. ZAHARIM, A. and SOPIAN, K. 2011. The effects of drying air 103(3), 900–905. temperature and humidity on the drying kinetics of seaweed. LARRAURI, J.A., RUPEREZ, P. and SAURA-CALIXTO, F. 1997. In Recent Researches in Geography, Geology, Energy, Effect of drying temperature on the stability of polyphenols Environment and Biomedicine, pp. 129–133, World Scientific and antioxidant activity of red grape pomace peels. J. Agric. and Engineering Academy and Society (Wseas) LLC, Bulgaria. Food Chem. 45(4), 1390–1393. GAN, C.-Y. and LATIFF, A.A. 2011. Optimisation of the solvent LE LANN, K., JEGOU, C. and STIGER-POUVREAU, V. 2008. extraction of bioactive compounds from Parkia speciosa pod Effect of different conditioning treatments on total phenolic using response surface methodology. Food Chem. 124(3), content and antioxidant activities in two Sargassacean species: 1277–1283. Comparison of the frondose Sargassum muticum (Yendo) GANESAN, P., MATSUBARA, K., OHKUBO, T., TANAKA, Y., Fensholt and the cylindrical Bifurcaria bifurcata R. Ross. NODA, K., SUGAWARA, T. and HIRATA, T. 2010. Phycol. Res. 56(4), 238–245. Anti-angiogenic effect of siphonaxanthin from green alga, LI, B., SMITH, B. and HOSSAIN, M.M. 2006. Extraction of Codium fragile. Phytomedicine 17(14), 1140–1144. phenolics from citrus peels: I. Solvent extraction method. GUERRA DORE, C.M., FAUSTINO ALVES, M.G., SANTOS, Separation Purification Technol. 48(2), 182–188. N.D., CRUZ, A.K., CAMARA,^ R.B., CASTRO, A.J., LIJUN, H., NIANJUN, X., JIANGONG, S., XIAOJUN, Y. and GUIMARAES~ ALVES, L., NADER, H.B. and LEITE, E.L. 2013. CHENGKUI, Z. 2005. Isolation and pharmacological Antiangiogenic activity and direct antitumor effect from a activities of bromophenols fromRhodomela confervoides. sulfated polysaccharide isolated from seaweed. Microvasc. Chin. J. Oceanol. Limnol. 23(2), 226–229. Res. 88, 12–18. LIM, Y. and MURTIJAYA, J. 2007. Antioxidant properties of GUPTA, S. and ABU-GHANNAM, N. 2011. Bioactive potential Phyllanthus amarus extracts as affected by different drying and possible health effects of edible brown seaweeds. Trends methods. LWT – Food Sci. Technol. 40(9), 1664–1669. Food Sci. Technol. 22(6), 315–326. MILLAR, A. and KRAFT, G. 1994. Catalogue of marine brown GUPTA, S., COX, S. and ABU-GHANNAM, N. 2011. Effect of algae (Phaeophyta) of New South Wales, including Lord different drying temperatures on the moisture and Howe Island, south-western Pacific. Aust. Syst. Bot. 7(1), phytochemical constituents of edible Irish brown seaweed. 1–47. LWT – Food Sci. Technol. 44(5), 1266–1272. MOHD ZAINOL, M., ABDUL-HAMID, A., ABU BAKAR, F. HASSAN KHAN, M.T. and ATHER, A. 2007. Potentials of and PAK DEK, S. 2009. Effect of different drying methods on phenolic molecules of natural origin and their derivatives as the degradation of selected flavonoids in Centella asiatica. Int. anti-HIV agents. Biotechnol. Annu. Rev. 13, 223–264. Food Res. J. 16, 531–537.

10 of 11 Journal of Food Processing and Preservation 2017; 41: e13025; VC 2016 Wiley Periodicals, Inc. T.T. DANG ET AL. THE EFFECT OF DRYING ON PROPERTIES OF THE ALGA

PISOSCHI, A.M. 2011. Methods for total antioxidant activity Influence of hot-air temperature on drying kinetics, determination: A review. Biochem. Anal. Biochem. 1, 106. functional properties, colour, phycobiliproteins, antioxidant PRIOR, R.L., WU, X. and SCHAICH, K. 2005. Standardized capacity, texture and agar yield of alga Gracilaria chilensis. methods for the determination of antioxidant capacity and LWT – Food Sci. Technol. 44(10), 2112–2118. phenolics in foods and dietary supplements. J. Agric. Food THAIPONG, K., BOONPRAKOB, U., CROSBY, K., Chem. 53(10), 4290–4302. CISNEROS-ZEVALLOS, L. and HAWKINS BYRNE, D. 2006. RAJAURIA, G., JAISWAL, A.K., ABU-GHANNAM, N. and Comparison of ABTS, DPPH, FRAP and ORAC assays for GUPTA, S. 2010. Effect of hydrothermal processing on colour, estimating antioxidant activity from guava fruit extracts. antioxidant and free radical scavenging capacities of edible J. Food Compos. Anal. 19(6), 669–675. Irish brown seaweeds. Int. J. Food Sci. Technol. 45(12), TOONTOM, N., MEENUNE, M., POSRI, W. and LERTSIRI, S. 2485–2493. 2012. Effect of drying method on physical and chemical SHAHIDI, F., JANITHA, P. and WANASUNDARA, P. 1992. quality, hotness and volatile flavour characteristics of dried Phenolic antioxidants. Crit. Rev. Food Sci. Nutr. 32(1), chilli. Int. Food Res. J. 19(3), 1023–1031. 67–103. VUONG, Q.V., GOLDING, J.B., NGUYEN, M.H. and ROACH, SHEIH, I.-C., FANG, T.J., WU, T.-K. and LIN, P.-H. 2009. P.D. 2013. Preparation of decaffeinated and high caffeine Anticancer and antioxidant activities of the peptide fraction powders from green tea. Powder Technol. 233, 169–175. from algae protein waste. J. Agric. Food Chem. 58(2), WONG, K. and CHEUNG, P.C. 2001. Influence of drying 1202–1207. treatment on three Sargassum species 2. Protein extractability, SITHRANGA BOOPATHY, N. and KATHIRESAN, K. 2011. in vitro protein digestibility and amino acid profile of protein Anticancer drugs from marine flora: An overview. J. Oncol. concentrates. J. Appl. Phycol. 13(1), 51–58. 2010, Article ID 214186. WU, S., LI, F., JIA, S., REN, H., GONG, G., WANG, Y. and LV, Z. SUN, Y., SHEN, Y., LIU, D. and YE, X. 2015. Effects of drying 2014. Drying effects on the antioxidant properties of methods on phytochemical compounds and antioxidant polysaccharides obtained from Agaricus blazei Murrill. activity of physiologically dropped un-matured citrus fruits. Carbohydr. Polym. 103, 414–417. LWT – Food Sci. Technol. 60(2), 1269–1275. ZHENG, M., XIA, Q. and LU, S. 2015. Study on drying methods TELLO-IRELAND, C., LEMUS-MONDACA, R., and their influences on effective components of loquat flower VEGA-GALVEZ, A., LOPEZ, J. and DI SCALA, K. 2011. tea. LWT – Food Sci. Technol. 63(1), 14–20.

Journal of Food Processing and Preservation 2017; 41: e13025; VC 2016 Wiley Periodicals, Inc. 11 of 11 Paper III:

“Optimisation of ultrasound-assisted extraction conditions for phenolic content and antioxidant activities of the alga Hormosira banksii using response surface methodology”. Journal of Applied Phycology 29(6): 3161-3173. doi.org/10.1007/s10811-017-1162-y.

J Appl Phycol DOI 10.1007/s10811-017-1162-y

Optimisation of ultrasound-assisted extraction conditions for phenolic content and antioxidant activities of the alga Hormosira banksii using response surface methodology

Thanh T. Dang1,2 & Quan Van Vuong1 & Maria J. Schreider1 & Michael C. Bowyer1 & Ian A. Van Altena1 & Christopher J. Scarlett1,3

Received: 1 October 2016 /Revised and accepted: 2 May 2017 # Springer Science+Business Media Dordrecht 2017

Abstract This study aimed to optimise ultrasound-assisted using RSM is effective for extraction and further isolation extraction (UAE) conditions of the brown alga Hormosira and purification of phenolic compounds from H. banksii.In banksii for total phenolic content (TPC) and antioxidant ac- addition, this alga could be a potential rich source of natural tivities including total antioxidant capacity (ABTS), DPPH antioxidants applied in food and pharmaceutical fields. free radical scavenging capacity (DPPH) and ferric reducing antioxidant power (FRAP) using response surface methodol- Keywords Hormosira banksii . Phaeophyceae . ogy (RSM). Box–Behnken design was employed to assess the Ultrasonic-assisted extraction (UAE) . Optimisation . RSM effect of ultrasonic temperature, time and power on the TPC and antioxidant activities of the extracts. The results showed that RSM was an accurate and reliable method in predicting Introduction TPC and antioxidant activities (ABTS, DPPH and FRAP) of the extracts with R2 values of 0.97, 0.96, 0.92 and 0.94, re- Hormosira banksii (Turner) Decaisne, a brown alga (Fucales: spectively. The ultrasonic temperature and time had the sig- Phaeophyta), is widely distributed along the coastal areas of nificant impact on TPC and antioxidant capacities. The opti- New South Wales, Australia (Millar and Kraft 1994). In recent mal UAE conditions for the maximal values of TPC and an- years, marine resources have attracted attention in the search tioxidant activities were of 30 °C, 60 min and power 60%, or for natural products in order to develop new drugs and healthy 150 W. The values of TPC and antioxidant activities (ABTS, foods (El Gamal 2010; Mohamed et al. 2012) and marine DPPH, FRAP) achieved under these parameters were 23.12 algae have been reported as a potential source for the extrac- − − − (mg GAE g 1), 85.64 (mg TE g 1), 47.24 (mg TE g 1)and tion of bioactive compounds (Gupta and Abu-Ghannam − 12.56 (mg TE g 1), respectively. UAE was found to be more 2011). Many metabolites have been isolated from brown al- efficient in comparison to conventional extraction, with gae, including pigments (fucoxanthin, carotenoids, etc.), phe- shorter time for extraction and higher of TPC level and anti- nolic compounds (phlorotannins), sulfated polysaccharides oxidant activities. Therefore, ultrasonic-assisted extraction (fucoidan), bromophenols and meroditerpenoids. These bio- active compounds have been linked to various health benefits such as being anti-allergic (Miyake et al. 2006), preventing * Christopher J. Scarlett cardiovascular diseases (Shi et al. 2010), neuroprotective ef- [email protected] fects (Alghazwi et al. 2016), anti-diabetes (Okada et al. 2004), and against several types of cancers (Cabrita et al. 2010;De 1 School of Environmental and Life Sciences, Faculty of Science and Information Technology, University of Newcastle, Ourimbah, NSW, Souza et al. 2009;Ferreresetal.2012; Kumar et al. 2013; Australia Kwak 2014). 2 Department of Seafood Processing Technology, Faculty of Food Recently, conventional extraction was considered to have Technology, Nha Trang University, Nha Trang, Khanh Hoa, Vietnam some drawbacks due to long time, high cost and degradation 3 Nutrition Food & Health Research Group, School of Environmental of product quality, while using organic solvents has to be and Life Sciences, University of Newcastle, Brush Rd, minimised for extraction because of potential health and en- Ourimbah, NSW 2258, Australia vironmental concerns (Azmir et al. 2013; Polshettiwar and J Appl Phycol

Varma 2008). Until now, various novel techniques have been steel mesh sieve (Endecotts Ltd., England) and stored developed for extracting components from algae (Kadam et al. at −20 °C for further analysis. 2013) such as pressurised liquid extraction (PLE) (Onofrejová et al. 2010), supercritical fluid extraction (SFE) (Tanniou et al. Ultrasound-assisted extraction (UAE) 2013), enzyme-assisted extraction (EAE) (Wijesinghe and Jeon 2012) and ultrasound-assisted extraction (UAE) The freeze-dried alga was extracted using ethanol 70% (Roselló-Soto et al. 2015). UAE is considered an effective (v/v) with a solvent to material ratio of 50 (mL g−1). method in comparison with the others due to its low energy UAE was conducted using an ultrasonic bath (Soniclean, requirements and low solvent consumption (Chemat and 220 V, 50 Hz and 250 W, Soniclean Pty Ltd., Australia). Khan 2011). Ultrasound enhances extraction efficacy by using The experimental parameters applied for extracting were an ultrasonic wave that helps increase penetration of solvent designed by response surface methodology (RSM) (JMP into the materials and the contact surface area between solid software, version 13). After ultrasonic extraction, the ex- and liquid phases. Moreover, the improvement in the extrac- tracts were immediately cooled on ice to room tempera- tion process using ultrasound is related to the destruction of ture (RT), filtered using a 0.45-μm cellulose syringe filter the cell walls, reduction of the particle size and enhancement (Phenomenex Australia Pty. Ltd., Australia) and diluted to of the mass transfer through the cell wall due to the collapse of the required volume for quantitative analysis. bubbles produced by cavitation (Wang et al. 2008;Macías- Sánchez et al. 2009; Teh and Birch 2014). The advantages and drawbacks of UAE from plants have been highlighted in pre- Conventional extraction vious studies (Romanik et al. 2007; Chemat and Khan 2011). Response surface methodology (RSM) is a useful tech- Conventional extraction was conducted according to the nique for optimising processes or products by establishing a method of Hossain et al. (2012) with some modifications. statistical and mathematical model. This model allows the The dried and ground sample (0.5 g) was extracted with ethanol (70%) and the ratio of solvent to material evaluation of multiple parameters and their interactions using −1 quantitative data, leading to a reduction in the number of ex- (50/1 mL g ) at 30 °C. The samples were shaken for perimental trials required (Pompeu et al. 2009;Wuetal. 12 h using a shaking water bath. The extracts were then 2012). Thus, it is also faster and more economical than other immediately cooled on ice to RT, filtered and diluted to approaches required for an optimisation process. the required volume for analysis. This study aimed to optimise the ultrasonic conditions for extraction of total phenolic content (TPC) and antioxidant Response surface methodology (RSM) activities of the H. banksii using RSM. Conventional extrac- tion was also applied to compare its effectiveness with the RSM with a Box-Behnken design was employed for designing optimum ultrasonic extraction. The findings of this study have experimental conditions to determine the influence of the three potential to be applied for further isolation and purification of independent parameters including ultrasonic temperature, time phenolic compounds from H. banksii. and power on the TPC and antioxidant activities of the extracts. The optimal ranges of temperature (30–50 °C), time (20–60 min) and power of ultrasound (60–100% or 150–250 W) were deter- Materials and methods mined based on preliminary experiments (data not shown). The independent variables and their code variable levels are shown in Materials Table 1. To express the TPC value or antioxidant activities as a function of the independent variables, a second-order polynomial The brown alga H. banksii was collected in March 2016 equation (Eq. 1) was used as follows and previously described by from Bateau Bay rocky shore, NSW, Australia. After Vuong et al. (2014): collection, the sample was washed with seawater to re- ¼ β þ ∑k β þ ∑k−1 ∑k β þ ∑k β 2 ð Þ move natural residues (sand and epifauna), kept in a Y 0 i¼1 iX i i¼1 j¼2 ijX iX j i¼1 ijX ij 1 box to protect from light and immediately transported i< j to the laboratory. The alga was then washed thoroughly with fresh water, immersed in liquid nitrogen, and freeze-dried for 48 h using a freeze dryer (Thomas where various Xi values are independent variables af- Australia Pty. Ltd., Australia) with a drying chamber fecting the responses Y; β0, βi, βii and βij are the re- pressure of 2 × 10−1 mbar and a cryo-temperature of gression coefficients for intercept, linear, quadratic and −50 °C. The dried sample was ground to give ≤0.6- interaction terms, respectively; and k is the number of mm particle size using a 0.6-mm EFL 2000 stainless variables. The three independent ultrasonic parameters J Appl Phycol

Table 1 The independent variables and their levels used for Box- (DPPH) assay as described by Brand-Williams et al. (1995). Behnken design Methanol and trolox were used as a control and standard for

Xi Factor levels the assay. The standard curve was linear between 25 and 1000 μM trolox. Results were expressed in mg TE g−1 dried −10 +1material.

Temperature (X1) (°C) 30 40 50 Ferric reducing antioxidant power (FRAP) The extract was Time (X ) (min) 20 40 60 2 diluted and its iron chelating capacity was analysed using the Power of ultrasound (X ) (%) 60 80 100 3 FRAP assay as described by Benzie and Strain (1999). Trolox was used as the calibration standard. The standard curve was linear between 25 and 1000 μM trolox, and the results were −1 were assigned as X1 (temperature, °C), X2 (time, min) expressed in mg TE g dried material. and X3 (% ultrasonic power). Thus, the function con- taining these three independent variables was expressed Statistical analysis as follows (Eq. 2): RSM experimental design and analysis were conducted Y ¼ β þ β X þ β X þ β X þ β X X 0 1 1 2 2 3 3 12 1 2 using JMP software (version 13). The software was also þ β þ β þ β 2 þ β 2 used to establish the model equation to graph the 3D- 13X 1X 3 23X 2X 3 11X 1 22X 2 and 2D contour plots of variable response and to predict þ β 2 ð Þ 33X 3 2 optimum values for the three response variables. All measurements were taken in triplicate (n = 3). Results were expressed as mean values with standard deviations, Determination of total phenolic content and significant differences between treatments were test- ed using analysis of variance and the LSD post hoc test Total phenolic content (TPC) The extract was diluted to fit with a 95% significance level (P < 0.05). Correlations within the optimal absorbance range for colorimetric assess- among data were calculated using Pearson’s correlation ment. TPC was determined according to the method of Vuong coefficient and expressed as R2. The SPSS version 16.0 et al. (2013). Briefly, 0.5 mL of diluted sample, 2.5 mL of 10% statistical package was used for all analyses. (v/v) Folin–Ciocalteu reagent was added, followed by the ad- dition of 2 mL of NaCO3 7.5% (w/v), then mixed well on a vortex vibrator for 2 min and incubated in the dark at room Results temperature (RT) for 1 h before the absorbance was measured at 765 nm. Gallic acid was used as the standard for the con- Fitting of the models struction of a calibration curve, with the results expressed as mg of gallic acid equivalents per gram of dried material (mg Fitting the models for the TPC values and antioxidant −1 GAE g ). activities is important to assess how precisely the RSM mathematical model can predict the ideal variances and Determination of antioxidant activities determine the correlations of the selected parameters to ultrasonic extractions. Therefore, analysis of variance ABTS total antioxidant capacity (ABTS) The extract was was undertaken to evaluate the reliability of the RSM diluted to fit within the optimal absorbance range for mathematical model. The TPC values and antioxidant ac- colorimetric assessment. Total antioxidant capacity was tivities of the extract obtained from all the experiments measured using 2,2′-azino-bis-3-ethylbenzothiazoline-6- are presented in Table 2. Analysis of variance (ANOVA) sulphonic acid (ABTS) assay as described by Thaipong indicated that the coefficient of multiple determination et al. (2006). Methanol was used as control and trolox (R2) for the response of TPC was 0.97, suggesting that (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic ac- there was a close correlation between the actual and pre- id) was used as a standard. The standard curve was dicted values of the TPC model data. The value of lack of linear between 25 and 1000 μM trolox. Results were fit was used to determine the adequacy of the model and expressed as mg of trolox equivalents per gram of dried was not significant (P > 0.05), indicating that the model −1 material (mg TE g ). could adequately fit the experimental data (Table 3)(Wu et al. 2012). Furthermore, the P value and t ratio were DPPH free radical scavenging capacity (DPPH) The extract used to determine the significance of the coefficients of was diluted and analyzed using 1,1-diphenyl-2-picrylhydrazyl the quadratic polynomial models. The smaller P value and Table 2 Experimental (Exp.) and predicted (Pred.) values of total phenolic compounds (TPC) and antioxidant activities: total antioxidant capacity (ABTS), DPPH free radical scavenging capacity (DPPH) and ferric reducing antioxidant power (FRAP) of H. banksii obtained from Box-Behnken design

Ultrasonic conditions Antioxidant activity

−1 −1 −1 −1 Exp. run X1 (°C) X2 (min) X3 (%) TPC (mg g )ABTS(mgg)DPPH(mgg)FRAP(mgg)

Exp. Pred. Exp. Pred. Exp. Pred. Exp. Pred.

1 30 40 60 21.41 20.95 83.34 81.75 46.88 48.04 12.77 12.15 2 30 20 80 18.38 18.52 72.25 74.05 40.46 40.60 10.07 10.13 3 30 60 80 22.25 22.59 84.55 86.90 47.73 45.51 12.13 12.00 4 30 40 100 21.85 21.83 75.69 73.13 40.53 41.45 9.15 9.85 5 40 40 80 18.28 18.08 70.34 71.66 42.43 41.54 7.97 7.67 6 40 20 100 18.36 18.24 65.27 66.03 32.77 31.71 6.61 5.86 7 40 60 60 20.68 20.80 75.18 74.42 37.76 38.82 7.49 8.24 8 40 20 60 14.46 14.78 68.63 68.41 36.71 35.41 5.46 6.02 9 40 40 80 18.48 18.08 71.05 71.66 41.83 41.54 7.88 7.67 10 40 60 100 18.31 17.99 71.91 72.13 34.36 35.66 6.94 6.38 11 40 40 80 17.48 18.08 73.58 71.66 40.37 41.54 7.15 7.67 12 50 60 80 18.76 18.61 62.66 60.86 37.11 36.97 6.67 6.61 13 50 40 60 18.68 18.70 53.65 56.22 38.47 37.55 6.68 5.98 14 50 20 80 17.24 16.90 63.95 61.60 32.29 34.51 5.61 5.74 15 50 40 100 18.01 18.47 58.58 60.17 38.46 37.30 5.63 6.25 Valid. 30 60 60 23.12 ± 1.01a 24.07 ± 1.66a 85.64 ± 2.07a 88.97 ± 8.89a 47.24 ± 0.65a 46.44 ± 6.30a 12.56 ± 0.43a 13.02 ± 2.71a

In validation row (valid.), all the values are means ± standard deviations (n = 4) and the similar letters between Exp. and Pred. values are not significantly different (P > 0.05). The Pred. values were predicted by response surface methodology using JMP software (version 13) plPhycol Appl J J Appl Phycol

Table 3 Regression coefficients of the fitted quadratic equation for total phenolic compound (TPC) and antioxidant activities (ABTS, DPPH and FRAP)

TPC ABTS DPPH FRAP

Regression Regression t ratio Regression t ratio Regression t ratio Regression t ratio coefficients coefficient coefficient coefficient coefficient

β0 18.08 57.29 71.67 42.42 41.54 34.18 7.67 14.92 Linear

β1 −1.40*** −7.24 −9.62*** −9.30 −3.66** −4.92 −2.44*** −7.76

β2 1.45*** 7.48 3.02* 2.92 1.84 2.47 0.68 2.18

β3 0.16 0.84 −1.17 −1.13 −1.71 −2.30 −0.51 −1.62 Interaction

β12 −0.59 −2.15 −3.39 −2.32 −0.61 −0.58 −0.25 −0.56

β13 −0.28 −1.02 3.15 2.15 1.59 1.51 0.64 1.44

β23 −1.57** −5.74 0.02 0.02 0.14 0.13 −0.43 −0.96 Quadratic

β11 1.56** 5.47 −1.62 −1.06 1.77 1.62 1.44* 3.12

β22 −0.48 −1.68 0.81 0.53 −3.92* −3.57 −0.49 −1.06

β33 0.35 1.23 −2.22 −1.46 −2.22 −2.03 −0.55 -1.19 R2 0.97 0.96 0.92 0.94 P value of lack of 0.51 0.09 0.27 0.22 fit P values of 0.002 0.006 0.027 0.012 models

β0 is a constant; βi, βij and βii are the linear, interactive and quadratic coefficients of the second-order polynomial equation, respectively *, **, ***Significantly differences with P < 0.05, 0.01 and 0.001, respectively larger t ratio would indicate a more significant effect on ABTS, DPPH and FRAP, respectively (P > 0.05). the corresponding variables (Kha et al. 2013). The P val- Moreover, the P values of the models were 0.006, ue of the model was found to be 0.002 (showing the 0.027 and 0.012 and t ratio (42.42, 34.18 and 14.92) significance of the model considering a confidence inter- for different antioxidant activities (ABTS, DPPH and val of 99%) and the t ratio of the model was 57.29, re- FRAP, respectively) further confirming that there was a vealing that the mathematical model was reliable for strong reliability of these mathematical models in predicting TPC values, following the second-order poly- predicting the antioxidant activity for the following nomial formula (Eq. 3). second-order polynomial formulas (Eqs. (4), (5)and(6):

Y ABTS ¼ 71:67−9:62X 1 þ 3:02X 2−1:17X 3−3:39X 1X 2 Y ¼ 18:08−1:40X þ 1:45X TPC 1 2 þ : þ : − : 2 3 15X 1X 3 0 02X 2X 3 1 62X 1 þ 0:16X −0:59X X −0:28X X −1:57X X 3 1 2 1 3 2 3 þ : 2− : 2 ð Þ 0 81X 2 2 22X 3 4 þ : 2− : 2 þ : 2 ð Þ 1 56X 1 0 48X 2 0 35X 3 3

Y DPPH ¼ 41:54−3:66X 1 þ 1:84X 2−1:71X 3−0:61X 1X 2

þ 1:59X 1X 3 þ 0:14X 2X 3 Fitting of the models for the three different antioxi- þ : 2− : 2− : 2 ð Þ dant assays was also investigated. The results indicated 1 77X 1 3 92X 2 2 22X 3 5 that R2 values for the models of ABTS, DPPH and FRAP were 0.96, 0.92 and 0.94, respectively, revealing Y ¼ 7:67−2:44X þ 0:68X −0:51X −0:25X X the strong correlations between actual and predicted data FRAP 1 2 3 1 2

(Table 3). The values for lack of fit were 0.19, 0.15 and þ 0:64X 1X 3−0:43X 2X 3 0.15, indicating that there was no significant difference þ : 2− : 2− : 2 ð Þ between the predicted and experimental values for 1 44X 1 0 49X 2 0 55X 3 6 J Appl Phycol

−1 Fig. 1 The 3D response surface and 2D contour plots (a–c) of TPC (mg GAE g )ofH. banksii extract mutually affected by temperature (X1,°C),time (X2, min) and ultrasonic power (X3,%)

Effects of ultrasonic extraction conditions on the TPC TPC of extract when temperature decreased from 50 to 30 °C and ultrasonic time increased between 20 and The optimal levels of the independent variables for the 60 min at the constant power of 80%. Figure 1b shows that TPC were visualised by the 3D surface and 2D contour. TPC increased slightly when a decrease in temperature and The relationship between the independent variables and the an increase in level of ultrasonic power were applied at the responses was shown by response surface plots, while the constant time of 40 min. However, at the constant temper- contour plot indicated the shape of a response surface. The ature of 40 °C, the long time and low power or short time effect of the ultrasonic temperature, time and power on and high power both led to the high level of TPC of the TPC is presented in Table 3 by the coefficients of quadratic extract. The application of the long time and low power models. As the results, the two independent variables (ul- was found to be better for TPC (Fig. 1c). trasonic temperature and time) had significant impact on the TPC of H. banksii extract (P < 0.05). In addition, TPC Effects of ultrasonic extraction conditions on antioxidant was the most affected by ultrasonic time, followed by tem- activities perature and power of ultrasound. There was also the sig- nificant effect of the interaction (between independent var- Total antioxidant capacity (ABTS) iables: ultrasonic time × power) and quadratic term of tem- perature on the TPC of the algal extract (P < 0.01). From For ABTS, all three parameters of ultrasonic temperature, time Fig. 1a, it can be seen that there was a steady increase in and power affected ABTS values of the H. banksii extracts J Appl Phycol

−1 Fig. 2 The 3D response surface and 2D contour plots (a–c)ofABTS(mgTEg )ofH. banksii extract mutually affected by temperature (X1,°C),time (X2, min) and ultrasonic power (X3,%)

(Fig. 2a–c). The degree of the effect of the independent vari- The DPPH free radical scavenging capacity (DPPH) ables can be shown in the following order: temperature > time > power (based on the P values). The statistical results indi- Statistical results showed that only linearity of temperature cated that the ultrasonic temperature and time significantly and quadratic term of time significantly affected DPPH free affected the ABTS (P < 0.05, Table 3). In addition, Fig. 2a radical scavenging capacity of the extract (P <0.05;Table3). shows that ABTS values of H. banksii rapidly decreased when The order of the influence of the parameters on the values of the temperature increased between 30 and 50 °C, while in- DPPH was temperature > time > power. Figure 3a illustrates creasing moderately with ultrasonic time from 20 to 60 min that the DPPH value of H. banksii extract rose steadily when at the constant power of 80%. A decrease in temperature and ultrasonic temperature went down from 50 to 30 °C and the power resulted in the increase of ABTS in the algal extract at ultrasonic time increased from 20 and 46.15 min at the power the constant time of 40 min (Fig. 2b). At the constant temper- 80%. High DPPH was also observed at low temperature ature of 40 °C, high ABTS was also observed when long time (30 °C) and power (60% or 150 W) at the constant time of and low power of ultrasound was applied (Fig. 2c). Therefore, 40 min (Fig. 3b). With the temperature at 40 °C, the moderate it was found that the highest value of ABTS (88.97 mg time (about 46 min) and low power resulted in the high value TE g−1)fromH. banksii extract could be obtained at the op- of DPPH of the extract (Fig. 3c). From the results, the ultra- timal ultrasonic parameters (temperature of 30 °C, time of sonic parameters affected DPPH had the similar trend to 60 min and power of 60%). ABTS of the extract except for a decrease of DPPH when time J Appl Phycol

−1 Fig. 3 The 3D response surface and 2D contour plots (a–c)ofDPPH(mgTEg )ofH. banksii extract mutually affected by temperature (X1,°C),time (X2, min) and ultrasonic power (X3,%) was from 46.15 to 60 min and the maximal value for DPPH predicted that the maximum value of FRAP (13.02 mg (48.38 mg TE g−1) could be obtained at the predicted condi- TE g−1) could be obtained with the conditions: ultrasonic tem- tions (temperature of 30 °C, time of 46.15 min and power of perature (30 °C), time (60 min) and the power (60% or 60%). 150 W).

Ferric reducing antioxidant power (FRAP) Optimisation and validation of UAE conditions for TPC and antioxidant activities Figure 4a–c and Table 3 outline the changes in FRAP of the extract under the different ultrasonic conditions. The FRAP The study aimed to determine the optimal conditions for TPC was significantly affected by the linearity of temperature and and antioxidant activities of the alga H. banksii using JMP 13 the quadratic term of temperature (P < 0.05). FRAP was most software. Through the process, the ultrasonic temperature, affected by temperature, followed by time and power of ultra- time and power were estimated to obtain the maximum values sound. An increase in the values of FRAP was observed as for TPC and antioxidant activities (ABTS, DPPH and FRAP). temperature decreased (between 50 and 30 °C) and time in- The results above and outlined in Fig. 5 indicated that the creased (between 20 and 60 min) at the moderate power of increase in temperature and power resulted in a decrease in 80% (Fig. 4a). The antioxidant activity of the extract increased TPC and antioxidant activities (except for the slight increase when both ultrasonic temperature and power decreased at the in TPC when power increased). A long time was also required time of 40 min (Fig. 4b). As shown in Fig. 4c, the long time for the high values of TPC and antioxidant activities (except and low power resulted in the high value of FRAP for the for the slight decrease in DPPH when time increased between extract at the temperature of 40 °C. From the model, it was 46.15 and 60 min). The theoretical maximum values of TPC, J Appl Phycol

−1 Fig. 4 The 3D response surface and 2D contour plots (a–c)ofFRAP(mgTEg )ofH. banksii extract mutually affected by temperature (X1,°C),time (X2, min) and ultrasonic power (X3,%)

Fig. 5 The predicted profilers of TPC and antioxidant activities at the condition, while blue lines indicate the 95% confidence intervals. The optimal conditions of the temperature (X1, °C), time (X2,min)and predicted profilers were predicted by response surface methodology ultrasonic power (X3,%).Solid lines indicate predicted mean values of using JMP software (version 13) TPC and antioxidant activities. Red dashed lines show the values at each J Appl Phycol

Table 4 The comparison between the UAE and conventional method for the conventional conditions, 12 h was required. This fur- Extraction methods ther confirmed the advantage of the ultrasonic method in term of the efficacy, quality of the extracts for extracting biologi- UAE Conventional extraction cally active compounds.

TPC (mg GAE g−1) 23.12 ± 1.01a 16.21 ± 0.56b −1 ABTS (mg TE g ) 85.64 ± 2.07a 51.32 ± 2.33b Discussion DPPH (mg TE g−1) 47.24 ± 0.65a 30.56 ± 2.26b −1 FRAP (mg TE g ) 12.56 ± 0.43a 8.34 ± 0.12b Figure 1a–c and Table 3 show the effect of extraction condi- All the values are means ± standard deviations (n = 4) and the different tions (temperature, time and power of ultrasound) on the re- letters in the same row are significantly different (P < 0.05) covery yield of phenolic compounds from H. banksii. The higher temperature created the higher solvent diffusion rate and mass transfer, while lower in the solvent viscosity and ABTS and FRAP could be obtained by combining the ultra- surface tension made more polyphenols dissolve into the ex- sonic temperature, time and power (Fig. 5). Based on the traction medium (Hossain et al. 2012), so the extraction yield prediction of the model, the highest TPC, ABTS and FRAP increased. However, there was the limitation in using high were 24.07 mg GAE g−1, 88.97 mg TE g−1 and 13.02 mg temperature for extracting active components of plants, espe- TE g−1, respectively, with the optimum conditions (tempera- cially algae, due to the effect of high temperature on thermo- ture of 30 °C, time of 60 min and power of 60%). The DPPH sensitive compounds (Shahidi et al. 1992; Le Lann et al. value was 46.44 mg TE g−1 with these parameters (gained 2008). On the other hand, it could be that a reduction of the 96% of the maximum value of DPPH). Therefore, the condi- cavitation by high temperature resulted in the decrease in ex- tions above were chosen as the optimal conditions for traction yield as well as TPC (Dey and Rathod 2013). It can be extracting the alga H. banksii. In addition, the experiments seen that low temperature was suitable for extracting phenolic were performed under these optimal conditions to validate compounds from H. banksii. the adequacy of the model prediction. The statistical results In terms of time for extracting, it was shown that there were showed that there was no significant difference between pre- two stages in the extraction process using UAE. The soluble dicted and measured responses of TPC and antioxidant activ- components on surfaces of the seaweed matrix were dissolved ities (ABTS, DPPH and FRAP) (P >0.05;Table2) and that in solvents (washing stage) and mass transfer of the solute the measured values of the responses were found to be well from the seaweed matrix into the solvent through diffusion fitted to the ones predicted by the regression model. and osmotic processes (slow extraction stage) (Kadam et al. Therefore, these conditions were suggested to extract the high 2015). Therefore, time was also required enough for solutes yield of TPC and antioxidant activities of H. banksii for fur- dissolving in solvents. Topuz et al. (2016) stated that the in- ther isolation and utilisation. In addition, these findings further crease in TPC value when the long time was applied for the confirmed the appropriateness of the models used for extraction of the red seaweed Laurencia obtusa with time optimising the extraction conditions using UAE technique. 58 min. However, time needed to be optimised due to the efficacy of extract and the degradation of phenolic Comparison of the extraction efficacy compounds in the extract. Han et al. (2011) showed that the between the conventional and UAE methods maximum ultrasound time for extracting phenolic compounds from Saccharina (Laminaria) japonica was 60 min, while Conventional extraction techniques are usually applied to ex- exceeding this ultrasonic time could lead to a decrease in tract bioactive compounds from the materials. Therefore, this TPC level due to degradation of bioactive compounds by ul- study conducted conventional extraction to compare the effi- trasonic wave. cacy versus ultrasonic extraction under the optimum condi- Regarding to ultrasonic power, previous studies also found tions. The results showed that the ultrasonic technique was that the application of higher ultrasonic power increased the effective to obtain significantly higher levels of TPC and an- recovery yield of TPC in different algal species. The increase tioxidant capacity when compared to the conventional one in TPC when higher ultrasonic power was applied could be (P < 0.05; Table 4). The level of TPC using the ultrasound explained by more damage to the cell wall under higher ultra- was 142.6% higher than the one by the conventional extrac- sonic power (Kadam et al. 2015). Furthermore, it is believed tion. In terms of antioxidant capacity, the values for ABTS, that ultrasonic power acts as a driving force for the dispersion DPPH and FRAP using the ultrasonic method were also of solvent into solid samples, resulting in the increase in ex- higher (166.8, 154.6 and 150.6%, respectively) in comparison traction yield (Han et al. 2011). However, this study found that with those of the conventional one. It should be noted that the there was only a slight increase in TPC of the extract when time applied for ultrasonic extraction was only 1 h, whereas higher ultrasonic power was applied. This could be because, J Appl Phycol as ultrasonic power was boosted, there was an increase in the compounds in algal extracts may also contribute to the anti- bubble numbers in the solvent during cavitation, leading to a oxidant properties (Matanjun et al. 2008). Other experiments reduction in the efficacy of ultrasound energy transmission are in progress for isolation and purification of bioactive com- into the medium (Filgueiras et al. 2000; Zhao et al. 2007) pounds and it was found that both pigments and phenolic and/or a decrease in TPC caused by ultrasonic wave with high compounds (phlorotannins) had significantly affected the an- power (Han et al. 2011; Teh and Birch 2014). The results from tioxidant activity of H. banksii extract. However, phenolic the model illustrated that critical values of temperature, time compounds showed the main role in antioxidant activity of and power of ultrasound for TPC of algal extraction were of this extract due to higher amount of phenolic compounds 30 °C, 60 min and 60%, respectively. At these conditions, the and antioxidant capacity in comparison to other compounds. TPC value obtained was about 24.07 mg GAE g−1. In conclusion, this study demonstrated that response sur- A rich source of antioxidants (phlorotannins, tocopherols, face methodology was appropriate for optimising the algal carotenoids, ascorbic acid, fatty acids, etc.) can be found in a extracts with the greatest of TPC and antioxidant activities wide range of algal species (Gupta and Abu-Ghannam 2011). of the alga H. banksii using ultrasonic-assisted extraction. In this study, antioxidant activity of the extract H. banksii was Ultrasonic temperature had the strongest influence on TPC evaluated by three simple, fast and reliable biochemical assays and antioxidant capacity, followed by ultrasonic time, and (ABTS, DPPH and FRAP) (Matanjun et al. 2008; Frankel and then power. The optimal UAE conditions for TPC yield and Meyer 2000; Tanniou et al. 2013). The results showed that the antioxidant capacity of H. banksii were temperature of 30 °C, highest ABTS, DPPH and FRAP of extract obtained with the time of 60 min and power of 60% (150 W). Under these low ultrasonic temperature and power (30 °C and 60%) and conditions, TPC and antioxidant activities (ABTS, DPPH, the long time (60 min), except for DPPH with the highest FRAP) achieved were 23.12 mg GAE g−1, 85.64 mg value at 46.15 min. TE g−1, 47.24 mg TE g−1, 12.56 mg TE g−1, respectively. There was a positive relationship between TPC and antiox- The extraction efficacy using UAE was significantly higher idant activities (ABTS, DPPH and FRAP) when the extracting compared to the conventional approach. From the findings, conditions changed (Fig. 5). This means that phenolic com- the conditions of extraction by UAE using RSM has potential pounds are the main contributors on the antioxidant activities to be applied for further isolation and purification of phenolic of this alga. It was confirmed by the strong correlations be- compounds from alga H. banksii and application of phenolic tween TPC and antioxidant activities (ABTS, DPPH and compounds of this alga in food and pharmaceutical fields. FRAP) of the extract H. banksii (r2 = 0.992, 0.993 and 0.920, respectively) in our previous study (Dang et al. Acknowledgements The authors would like acknowledge the follow- 2016). This finding is supported by Matanjun et al. (2008) ing funding support: Ramaciotti Foundation (ES2012/0104). The authors who indicated that the phenolic compounds mainly contribut- also kindly thank the University of Newcastle, Faculty of Science and IT, 2 the Vietnamese Government through the Vietnam International ed to FRAP of the methanolic extracts (r = 0.96). Previous Education Development - Ministry of Education and Training and the studies also illustrated the role of phenolic compounds in an- Ministry of Agriculture and Rural Development and the University of tioxidant activity based on the positive correlation between Newcastle for awarding a VIED-TUIT scholarship to Thanh Trung phenolic hydroxyl groups and DPPH free radical scavenge DANG. activity in several brown algae: Fucus vesiculocus, Laminaria ochroleuca, Sargassum muticum, Bifurcaria bifurcata, Laminaria sp. and Undaria pinnatifida (Jiménez- References Escrig et al. 2001;LeLannetal.2008;Amorimetal..2012). On the other hand, Charoensiddhi et al. (2015) reported that Alghazwi M, Kan YQ, Zhang W, Gai WP, Garson MJ, Smid S (2016) Neuroprotective activities of natural products from marine the low correlation between TPC and ORAC in Ecklonia macroalgae during 1999–2015. J Appl Phycol 28:3599–3616 radiata may imply that not only phlorotannins but also other Azmir J, Zaidul I, Rahman M, Sharif K, Mohamed A, Sahena F, Omar A organic compounds accounted for antioxidant activity of this (2013) Techniques for extraction of bioactive compounds from plant alga. Martins et al. (2013) reported that both Amansia sp. and materials: a review. J Food Eng 117:426–436 Cryptonemia seminervis presented the most relevant antioxi- Benzie I, Strain J (1999) Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modi- dant potential through DPPH assay, but they did not find any fied version for simultaneous measurement of total antioxidant pow- significant correlation between the antioxidant activity of the er and ascorbic acid concentration. Meth Enzymol 299:15–27 extracts and total phenolic content. In addition, it was found Brand-Williams W, Cuvelier M, Berset C (1995) Use of a free radical that antioxidant activity from Spatoglossum schroederi was method to evaluate antioxidant activity. LWT-Food Sci Technol 28: – mainly related to the present of flavonoids (Júnior et al. 25 30 Cabrita MT, Vale C, Rauter AP (2010) Halogenated compounds from 2015). It can be seen that except for phenolic compounds, marine algae. Mar Drugs 8:2301–2317 some other active compounds such as pigments (carotenoids, Charoensiddhi S, Franco C, Su P, Zhang W (2015) Improved antioxidant fucoxanthin, etc.), fatty acid, vitamins A, E or some organic activities of brown seaweed Ecklonia radiata extracts prepared by J Appl Phycol

microwave-assisted enzymatic extraction. J Appl Phycol 27:2049– Sargassum muticum (Yendo) Fensholt and the cylindrical 2058 Bifurcaria bifurcata R. Ross. Phycol Res 56:238–245 Chemat F, Khan MK (2011) Applications of ultrasound in food technol- Macías-Sánchez M, Mantell C, Rodriguez M, de la Martínez de la Ossa ogy: processing, preservation and extraction. Ultrason Sonochem E, Lubián L, Montero O (2009) Comparison of supercritical fluid 18:813–835 and ultrasound-assisted extraction of carotenoids and chlorophyll a Dang TT, Vuong QV,Schreider MJ, Bowyer MC, Altena IAV,Scarlett CJ from Dunaliella salina. Talanta 77:948–952 (2016) The effects of drying on physico-chemical properties and Martins CDL, Ramlov F, Carneiro NPN, Gestinari LM, dos Santos BF, antioxidant capacity of the brown alga (Hormosira banksii Bento LM, Horta PA (2013) Antioxidant properties and total phe- (Turner) Decaisne). J Food Process Preserv. doi:10.1111/jfpp.13025 nolic contents of some tropical seaweeds of the Brazilian coast. J De Souza ÉT, Pereira de Lira D, Cavalcanti de Queiroz A, Costa da Silva Appl Phycol 25:1179–1187 DJ, Bezerra de Aquino A, Campessato Mella EA, de Oliveira Matanjun P, Mohamed S, Mustapha NM, Muhammad K, Ming CH Chaves MC (2009) The antinociceptive and anti-inflammatory ac- (2008) Antioxidant activities and phenolics content of eight species tivities of caulerpin, a bisindole alkaloid isolated from seaweeds of of seaweeds from north Borneo. J Appl Phycol 20:367–373 the genus Caulerpa.MarDrugs7:689–704 Millar A, Kraft G (1994) Catalogue of marine brown algae (Phaeophyta) Dey S, Rathod VK (2013) Ultrasound assisted extraction of β-carotene of New South Wales, including Lord Howe Island, south-western from Spirulina platensis. Ultrason Sonochem 20:271–276 Pacific. Aust Systemat Bot 7:1–47 El Gamal AA (2010) Biological importance of marine algae. Saudi Miyake Y, Sasaki S, Ohya Y, Miyamoto S, Matsunaga I, Yoshida T, Oda Pharmaceutical Journal 18:1–25 H (2006) Dietary intake of seaweed and minerals and prevalence of Ferreres F, Lopes G, Gil-Izquierdo A, Andrade PB, Sousa C, Mouga T, allergic rhinitis in Japanese pregnant females: baseline data from the Valentão P (2012) Phlorotannin extracts from Fucales characterized Osaka Maternal and Child Health Study. Ann Epidemiol 16:614– by HPLC-DAD-ESI-MSn: approaches to hyaluronidase inhibitory 621 capacity and antioxidant properties. Mar Drugs 10:2766–2781 Mohamed S, Hashim SN, Rahman HA (2012) Seaweeds: a sustainable Filgueiras A, Capelo J, Lavilla I, Bendicho C (2000) Comparison of functional food for complementary and alternative therapy. Trends ultrasound-assisted extraction and microwave-assisted digestion Food Sci Technol 23:83–96 for determination of magnesium, manganese and zinc in plant sam- Okada Y, Ishimaru A, Suzuki R, Okuyama T (2004) A new ples by flame atomic absorption spectrometry. Talanta 53:433–441 phloroglucinol derivative from the brown alga Eisenia bicyclis:po- Frankel EN, Meyer AS (2000) The problems of using one-dimensional tential for the effective treatment of diabetic complications. J Nat methods to evaluate multifunctional food and biological antioxi- Prod 67:103–105 dants. J Sci Food Agric 80:1925–1941 Onofrejová L, Vašíčková J, Klejdus B, Stratil P, Mišurcová L, Kráčmar S, Gupta S, Abu-Ghannam N (2011) Bioactive potential and possible health Vacek J (2010) Bioactive phenols in algae: the application of effects of edible brown seaweeds. Trends Food Sci Technol 22:315– pressurized-liquid and solid-phase extraction techniques. J 326 Pharmaceut Biomed Anal 51:464–470 Han D, Zhu T, Row KH (2011) Ultrasonic extraction of phenolic com- Polshettiwar V, Varma RS (2008) Aqueous microwave chemistry: a clean pounds from Laminaria japonica Aresch using ionic liquid as ex- and green synthetic tool for rapid drug discovery. Chem Soc Rev 37: traction solvent. Bull Kor Chem Soc 32:2212–2216 1546–1557 Hossain MB, Brunton NP, Patras A, Tiwari B, O’Donnell C, Martin- Pompeu D, Silva E, Rogez H (2009) Optimisation of the solvent extrac- Diana AB, Barry-Ryan C (2012) Optimization of ultrasound assisted tion of phenolic antioxidants from fruits of Euterpe oleracea using extraction of antioxidant compounds from marjoram ( Origanum response surface methodology. Bioresour Technol 100:6076–6082 majorana L.) using response surface methodology. Ultrason Romanik G, Gilgenast E, Przyjazny A, Kamiński M (2007) Techniques Sonochem 19:582–590 of preparing plant material for chromatographic separation and anal- Jiménez-Escrig A, Jiménez-Jiménez I, Pulido R, Saura-Calixto F (2001) ysis. J Biochem Biophys Meth 70:253–261 Antioxidant activity of fresh and processed edible seaweeds. J Sci Roselló-Soto E, Galanakis CM, Brnčić M, Orlien V, Trujillo FJ, Mawson Food Agric 81:530–534 R, Barba FJ (2015) Clean recovery of antioxidant compounds from Júnior SQ, Carneiro VHA, Fontenelle TPC, de Sousa CL, Mesquita JX, plant foods, by-products and algae assisted by ultrasounds process- de Brito TV, Aragão KS (2015) Antioxidant and anti-inflammatory ing. Modeling approaches to optimize processing conditions. Trends activities of methanol extract and its fractions from the brown sea- Food Scie Technol 42:134–149 weed Spatoglossum schroederi. J Appl Phycol 27:2367–2376 Shahidi F, Janitha P, Wanasundara P (1992) Phenolic antioxidants. Crit Kadam SU, Tiwari BK, O’Connell S, O’Donnell CP (2015) Effect of Rev Food Sci Nutr 32:67–103 ultrasound pretreatment on the extraction kinetics of bioactives from Shi D, Li X, Li J, Guo S, Su H, Fan X (2010) Antithrombotic effects of brown seaweed (Ascophyllum nodosum). Separation Sci Technol bromophenol, an alga-derived thrombin inhibitor. Chin J Oceanol 50:670–675 Limnol 28:96–98 Kadam SU, Tiwari BK, O’Donnell CP (2013) Application of novel ex- Tanniou A, Leon ES, Laurent V, Elena I, Mendiola JA, Stéphane C, traction technologies for bioactives from marine algae. J Agric Food Valérie SP (2013) Green improved processes to extract bioactive Chem 61:4667–4675 phenolic compounds from brown macroalgae using Sargassum Kha TC, Nguyen MH, Phan DT, Roach PD, Stathopoulos CE (2013) muticum as model. Talanta 104:44–52 Optimisation of microwave-assisted extraction of Gac oil at different Teh SS, Birch EJ (2014) Effect of ultrasonic treatment on the polyphenol hydraulic pressure, microwave and steaming conditions. Int J Food content and antioxidant capacity of extract from defatted hemp, flax Sci Technol 48:1436–1444 and canola seed cakes. Ultrason Sonochem 21:346–353 Kumar SR, Hosokawa M, Miyashita K (2013) Fucoxanthin: a marine Thaipong K, Boonprakob U, Crosby K, Cisneros-Zevallos L, Hawkins carotenoid exerting anti-cancer effects by affecting multiple mecha- Byrne D (2006) Comparison of ABTS, DPPH, FRAP, and ORAC nisms. Mar Drugs 11:5130–5147 assays for estimating antioxidant activity from guava fruit extracts. J Kwak JY (2014) Fucoidan as a marine anticancer agent in preclinical Food Compos Anal 19:669–675 development. Mar Drugs 12:851–870 Topuz OK, Gokoglu N, Yerlikaya P, Ucak I, Gumus B (2016) Le Lann K, Jegou C, Stiger-Pouvreau V (2008) Effect of different con- Optimisation of antioxidant activity and phenolic compound extrac- ditioning treatments on total phenolic content and antioxidant activ- tion conditions from red seaweed (Laurencia obtusa). J Aquat Food ities in two Sargassacean species: comparison of the frondose Prod Technol 25:424–422 J Appl Phycol

Vuong QV, Goldsmith CD, Dang TT, Nguyen VT, Bhuyan DJ, Wijesinghe W, Jeon YJ (2012) Enzyme-assistant extraction (EAE) of Sadeqzadeh E, Bowyer MC (2014) Optimisation of ultrasound- bioactive components: a useful approach for recovery of industrially assisted extraction conditions for phenolic content and antioxidant important metabolites from seaweeds: a review. Fitoterapia 83:6–12 capacity from Euphorbia tirucalli using response surface methodol- Wu Y, Wang X, Fan E (2012) Optimisation of ultrasound-assisted extrac- ogy. Antioxidants 3:604–617 tion of puerarin and total isoflavones from Puerariae Lobatae Radix Vuong QV, Hirun S, Roach PD, Bowyer MC, Phillips PA, Scarlett CJ (Pueraria lobata (Wild.) Ohwi) with response surface methodology. (2013) Effect of extraction conditions on total phenolic compounds Phytochem Anal 23:513–519 and antioxidant activities of Carica papaya leaf aqueous extracts. J Zhao S, Kwok KC, Liang H (2007) Investigation on ultrasound assisted Herbal Med 3:104–111 extraction of saikosaponins from Radix bupleuri. Sep Purif Technol Wang J, Sun B, Cao Y, Tian Y, Li X (2008) Optimisation of ultrasound- 55:307–312 assisted extraction of phenolic compounds from wheat bran. Food Chem 106:804–810 Paper IV:

“Optimum conditions of microwave assisted extraction for phenolic compounds and antioxidant capacity of the brown alga Sargassum vestitum”. Separation Science and Technology (In Press). doi.org/10.1080/01496395.2017.1414845.

       

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Optimum conditions of microwave-assisted extraction for phenolic compounds and antioxidant capacity of the brown alga Sargassum vestitum Thanh T Danga,b, Michael C Bowyera, Ian A Van Altenaa, and Christopher J Scarletta aSchool of Environmental and Life Sciences, Faculty of Science and Information Technology, University of Newcastle, Ourimbah, New South Wales, Australia; bDepartment of Seafood Processing Technology, Faculty of Food Technology, Nha Trang University, Khanh Hoa, Nha Trang, Vietnam

ABSTRACT ARTICLE HISTORY This study aimed to optimise microwave-assisted extraction (MAE) conditions for total phenolic Received 13 June 2017 compounds (TPCs) and antioxidant activities of the alga Sargassum vestitum by using response Revised 4 December 2017 surface methodology with Box–Behnken design. The results showed that solvent concentration Accepted 5 December 2017 had the greatest impact on TPC and antioxidant activities of the extracts, followed by radiation KEYWORDS time and power. The optimal MAE conditions were ethanol concentration of 70%, radiation time Sargassum vestitum; brown of 75 s and power of 80%. The optimal MAE method showed much better extraction efficacy of algae; microwave-assisted phenolics and antioxidant capacities of the extract than conventional and ultrasonic methods. extraction (MAE); optimisation; RSM

Introduction problems.[8,9] Till date, various novel techniques have been developed for extracting components from algae Marine algae as potential renewable and sustainable such as pressurised liquid extraction, supercritical fluid sources could be exploited as the function ingredients extraction, enzyme-assisted extraction, ultrasound- for human health applications.[1] Among the marine assisted extraction (UAE), and microwave-assisted algae, the brown algae (Phaeophyceae) have shown to extraction (MAE).[7,10] Of these methods, MAE was possess higher antioxidant potential compared to red proved to be more advantageous compared to tradi- (Rhodophyceae) and green (Chlorophyceae) algae, and tional techniques due to the short time, selective heat- bioactive compounds not found in terrestrial sources.[2] ing, low consumption of solvents, high extraction Many metabolites isolated from the brown algae, efficacy and limitation in degradation of the target including pigments (fucoxanthin), phenolic com- components.[9] The reasons for these were because the pounds, sulfated polysaccharides (fucoidan), bromo- rapid internal heating process based on the interaction phenols and meroditerpenoids, have been linked to of the polar molecules with electromagnetic waves various health benefits such as being anti-allergic, pre- resulted in the damage of the plant tissues and/or cells.- venting cardiovascular diseases, anti-diabetes and [11] The target compounds from the ruptured plant cells against several types of cancers.[1,3,4] In brown algae, were rapidly dissolved in the solvents by high micro- phenolic compounds have a high ratio compared to the wave energy and low viscosity of solvents, leading to others and to be mainly responsible for antioxidant the high extraction efficacy. On the other hand, some activity of the extracts.[5,6] The structure of these com- disadvantages of MAE were outlined such as the degra- pounds varies from the simple molecules as phenolic dation of thermo-labile components due to the high acid to the complex compounds constructed by several power of microwave. However, there was no significant units of phloroglucinol monomer (1,3,5-trihydroxyben- difference for yield of flavonoids at the microwave zene) and called phlorotannins.[1] powder of 500–1000 W.[12] The advantages and draw- Recently, conventional extraction was considered to backs of MAE techniques used for plants have been also have some drawbacks because of long time, cost and summarised in previous studies.[7,11,13] degradation of the products[7], while using organic Response surface methodology (RSM) is a useful solvents has been minimised for extraction due to technique for optimising processes or products by potential health concerns and environmental establishing a statistical and mathematical model. This

CONTACT Christopher J. Scarlett [email protected] Deputy Head of School (Ourimbah) Head Discipline of Applied ScienceSchool of Environmental and Life Sciences, Head Pancreatic Cancer Research, University of Newcastle, 10 Chittaway Rd, Ourimbah, NSW 2258, Australia Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lsst. © 2017 Taylor & Francis 2 T. T. DANG ET AL. model allows the evaluation of multiple parameters and choose the optimal solvent for extraction of phenolic their interactions using quantitative data, leading to a compounds. reduction in the number of experimental trials required.[14] Thus, it is also faster and more economical Microwave-assisted extraction than other approaches required for an optimisation process. The freeze-dried alga (0.5 g) was extracted using etha- To date, the optimal extraction conditions for the nol 70% (v/v) with a solvent to material ratio of 50/1 − alga Sargassum vestitum using MAE have not been (mL⋅g 1). The process was conducted by using a micro- established. Therefore, this study aimed to optimise wave oven (Sharp Caurousel Inverter microwave the microwave conditions for extraction of the total 1200W, frequency 2450 MHz, Japan) in a fume hood. phenolic compound (TPC) value and antioxidant The sample was carried out as described by Alonso- capacity from the S. vestitum using RSM. Carrillo et al.[15] with slight modifications. After each Conventional extraction and UAE were also period of 5 s irradiation and power was switched off for employed to compare the extraction efficacy with cooling 10 s to avoid boiling out, and the sample was the optimal microwave extraction. The results shaken to increase the contact of the solid and solvent. showed that the extraction conditions for the extract The cooling system outside of the microwave oven S. vestitum were successfully optimised using RSM allows the continuous condensation of distillate. The (JMP 13 software). The extraction using MAE is measures ensured that the ratio of ethanol to water of better than the conventional and ultrasonic methods the solvent mixture was maintained through experi- relevant to the extraction efficacy and antioxidant ments. The parameters applied for extracting were activities of the extract. The findings of this study designed by RSM (JMP software, version 13). After have the potential to be applied for further isolation extraction, the extracts were immediately cooled on and purification of bioactive compounds of the alga ice to room temperature, filtered using a 0.45 μm cel- S. vestitum. lulose syringe filter (Phenomenex Australia Pty. Ltd., Lane Cove, Australia) and diluted to the required volume for quantitative analysis. Materials and methods

Materials Conventional and ultrasound-assisted extraction A brown alga S. vestitum was collected in March 2016 Conventional extraction and UAE were conducted from a rocky shore at Bateau Bay, NSW, Australia according to the methods of Hossain et al.[16] with ʹ ” ʹ ” (latitude of 33°22 55.2 S; longitude of 151°29 6 E). some modifications. The samples (0.5 g) were extracted After collection, the sample was washed with seawater using ethanol 70% with the ratio of solvent to material − to remove natural residues (sand and epifauna) and 50/1 (mL⋅g 1) at temperature of 30°C. The sample was washed thoroughly with freshwater, then freeze dried shaken for 12 h using a shaking water bath (model no. for 48 h using a freeze dryer (Thomas Australia Pvt. SWB 20, serial no. 404015143, Ratek instruments Pty. Ltd., Seven Hills, NSW, Australia). The dried sample Ltd, Australia) and UAE using an ultrasonic bath ≤ was ground to give 600 μm particle size using a sieve (Soniclean, 220 V, 50 Hz and 250 W, Soniclean Pty – (Endecotts Ltd., London, England) and stored at 20°C Ltd, Australia) with the following conditions: tempera- for further analysis. ture of 30°C, radiation time of 60 min and power of 250 W. Solvents for the extraction process Response surface methodology Several solvents were selected for extracting phenolic compounds including ethyl acetate (purity 99.8%), The important factors influenced on the yield of the acetone (99.5%), methanol (99.8%), ethanol (99.5%) TPC and antioxidant capacity of the extract were (Sigma-Aldrich, Australia) and mixtures of these sol- determined based on the preliminary experiments vents with water. The experiment was carried out using (data not shown). The irradiation time, ethanol per- a microwave with the conditions: the ratio of solvent to centage and microwave power were considered as the − material 50/1 (mL⋅g 1), the irradiation period of 5 s for main factors for the optimal procedure. RSM with a total 60 s of the irradiation treatment and microwave Box–Behnken design was employed to determine the power of 80% (960 W). The extracts were evaluated for effect of the three independent parameters on the the recovery yield of TPC and antioxidant activities to TPC and the activity of the extract. The optimal SEPARATION SCIENCE AND TECHNOLOGY 3 ranges of irradiation time (25–75 s), ethanol percen- DPPH: The extract was analysed as described by tage (30–70%) and power of microwave (60–100% or Brand-Williams et al.[19] DPPH assay was used for 720–1200 W) were determined, and the independent testing free radical scavenging activities of the extracts. variables and their code variable levels are shown in Brand-Williams et al.[19] reported that the DPPH radi- Table 2. A second-order polynomial equation was cal scavenging capacity of components depended on used to express TPC value and antioxidant activities their ability to pair off with the unpaired electron of a (total antioxidant capacity (ABTS), free radical radical. Trolox was used as the standard, and the colour scavenging capacity (DPPH) and ferric-reducing anti- of the sample was read at 515 nm. The results were − oxidant power (FRAP)) as a function of the indepen- expressed as mg TE⋅g 1 dried alga. dent variables as follows (Eq. 1): FRAP: The extract was carried out as described by Benzie and Strain.[20] The FRAP assay measures the Y ¼ β þ β X þ β X þ β X þ β X X þ β X X 0 1 1 2 2 3 3 12 1 2 13 1 3 ability of an antioxidant compound to reduce a ferric þ β þ β 2 þ β 2 þ β 2 3+ 2+ 23X2X3 11X1 22X2 33X3 oxidant (Fe ) to a ferrous complex (Fe ) by electron- (1) transfer, which indicates the capacity of the compound to reduce reactive species.[20] Trolox was used as a β β β β where Y indicates the response variables; 0, i, ij and ii standard, and the sample was measured at 593 nm. − are the constant, linear, interactive and quadratic coeffi- The results were expressed as mg TE⋅g 1 dried alga. cients, respectively; Xi values are the levels of the inde- pendent variables as X1 (irradiation time, s), X2 (ethanol percentage, %) and X3 (power of microwave, %). Statistical analysis RSM experimental design and analysis were conducted Determination of total phenolic content using a JMP software. The software was also used to establish the model equation to graph the 3D- and 2D- Total phenolic content (TPC) was conducted as contour plots of variable response and to predict the described by Skerget et al.[17] with slightly modifica- optimum values for the three response variables. tions. Briefly, 0.5 mL of diluted sample, 2.5 mL of 10% Results were expressed as mean values with standard (v/v) Folin–Ciocalteu reagent was added, followed by deviations (n = 3), and significant differences between the addition of 2 mL of Na CO 7.5% (w/v), then 2 3 treatments were tested using analysis of variance and mixed well on a vortex vibrator for 2 min and incu- the the least significant difference (LSD) post-hoc test bated in the dark at room temperature for 1 h before for yields and antioxidant activities of the extracts by the absorbance was measured at 765 nm using a UV solvents and methods with a 95% significance level spectrophotometer (Varian Australia Pty. Ltd., (P < 0.05). Correlations among data were calculated Victoria, Australia). Gallic acid was used as a standard, using Pearson’s correlation coefficient and expressed and the results were expressed as milligram of gallic 2 as R . The SPSS software version 16.0 statistical package acid equivalents per gram of the dried alga (mg − (SPSS Inc; Chicago, IL, USA) was used for all analyses. GAE⋅g 1).

Results and discussion Determination of antioxidant capacity Solvents for extraction ABTS was measured as described by Thaipong et al.[18] ABTS assay based on the scavenging ability of antiox- The impact of different solvents on the recovery yield idants to the radical anion ABTS*+ was used to deter- of TPC and antioxidant capacity of the extract is shown mine antioxidant activity of algal extract. The radical in Table 1. It can be seen that the mixture of acetone anion ABTS*+ is generated by reacting a strong oxidis- and water (7/3 v/v) was the best solvent for extracting ing (potassium persulfate) with an ABTS salt. the components from the S. vestitum extract with the − Reduction of blue-green ABTS*+ radical coloured solu- higher TPC value (74.05 mg GAE⋅g 1) and antioxidant tion by hydrogen-donating antioxidant is measured.[18] activities (ABTS, 167.42; DPPH, 142.40; and FRAP, − Methanol and trolox (6-hydroxy-2,5,7,8- tetramethyl- 107.28 mg TE⋅g 1) of the extract compared to the rest chroman-2-carboxylic acid) were used as a control and of the solvents tested. The mixture of methanol or a standard, respectively, and the absorbance was mea- ethanol with water were also indicated as the potential sured at 734 nm. The results were expressed as milli- solvents for extraction. It was noted that the pure gram of trolox equivalents per gram of dried alga (mg organic solvents were not effective for extraction of − TE⋅g 1). the phenolic compounds in our study. TPC values 4 T. T. DANG ET AL.

Table 1. Effects of solvents to the recovery yield of TPC and antioxidant activities of the extract. Antioxidant assays Solvents TPC (mg GAE⋅g−1) ABTS (mg TE⋅g−1) DPPH (mg TE⋅g−1) FRAP (mg TE⋅g−1) Ethyl acetate 18.77 ± 1.86c 64.48 ± 1.58d 33.83 ± 5.48c 30.35 ± 3.29c Acetone 10.84 ± 0.93a 20.01 ± 1.42a 17.63 ± 1.02a 9.46 ± 0.99a Acetone 30% 60.73 ± 0.92g 132.68 ± 2.43h 109.72 ± 4.18k 90.07 ± 2.76g Acetone 70% 74.05 ± 1.39h 167.42 ± 3.18k 142.40 ± 1.07j 107.28 ± 4.29h Methanol 18.68 ± 0.29c 52.69 ± 0.66c 22.36 ± 0.28b 27.30 ± 1.01c Methanol 30% 50.98 ± 1.28f 114.23 ± 2.25f 75.62 ± 3.99e 52.2 ± 2.19e Methanol 70% 59.13 ± 1.27g 134.62 ± 2.80h 96.43 ± 4.71g 70.8 ± 5.36f Ethanol 14.87 ± 0.16b 40.7 ± 1.61b 23.66 ± 1.15b 19.49 ± 0.97b Ethanol 30% 46.92 ± 1.26e 97.67 ± 2.53e 65.88 ± 2.41d 55.29 ± 6.66e Ethanol 70% 51.65 ± 1.18f 126.91 ± 2.18g 84.77 ± 1.99f 73.14 ± 3.7f Water 33.74 ± 0.49d 65.48 ± 3.18d 39.83 ± 2.41c 40.65 ± 3.32d All the values are means ± standard deviations (n = 3), and the different letters in the same column are significantly different (P<0.05). obtained from these solvents were lower than 20 mg methanol were better than of ethanol for the extraction. − GAE⋅g 1, while it was significantly higher (33.74 It was in line with Wang et al.,[6] who reported that the − GAE⋅g 1) as water applied. The higher ratio of solvent highest TPC was found in alga Fucus vesiculosus to water was used, the higher recovery efficacy of TPC Linnaeus using acetone 70% as a solvent, while high and antioxidant capacity was gained. In other words, TPC was also found with methanol. It is explained that the higher values of TPC and antioxidant activities were protein–polyphenol complex formation may be inhib- observed in the mixtures of the solvents acetone, ited or hydrogen bonds between phenolic group and methanol and ethanol with water at the ratio of 7/3 protein carboxyl group can be broken down by acetone (v/v) compared to the ratio of 3/7 (v/v) or the pure during extraction. However, both acetone and metha- solvents (Table 1). It could be due to the increase in nol had high toxicity and not practical in food and swelling of the samples by water led to the increase of pharmaceutical fields. In addition, high extraction effi- the contact surface between the material matrix and the cacy of TPC and antioxidant activities of the extracts solvent.[21] It also made the bonds between components using the mixture of ethanol and water was observed with the wall of the material cells become weak that compared to the mixtures of acetone and methanol enhanced efficacy of the compounds dissolving in the with water (Table 1). Thus, the mixture of ethanol solvents. With regard to concentration of the organic and water was required as the solvent for the extraction solvent, the higher solvent percentage was applied, the of the constituents of the S. vestitum extract. yield of TPC and antioxidant capacity increased by the high proportion of the solvent dissolving the phenolic Fitting of the models compounds. In addition, the high solvent percentage resulted in more microwave energy absorbed and trans- The TPC values and antioxidant activities of the extract formed into heat in the sample that made the increase obtained from all the experiments are presented in of the kinetic of extraction as well as the decrease in the Table 3. In order to ensure that the RSM mathematical viscosity of the extraction media.[22] These factors models are reliable for predicting the ideal variances raised recovery yield of phenolic compounds as well and determining correlations of the selected parameters as the activity of the extracts (Table 2). to the extraction process, analysis of variance was iden- The selection of the solvent suitable for extracting tified and examined (Table 4). The results indicated phenolic compounds of the extracts was a necessary that the coefficient of multiple determination (R2) for step in developing extraction method. The chosen sol- the response of TPC was 0.88, suggesting that there was vents had to be costly and less toxic that was safe to a close correlation between the actual and predicted human and the environment. From the findings, it was values of the TPC model data. The value of lack of fit shown that the mixtures with water of acetone and was used to determine the adequacy of the model and was not significant (P > 0.05), suggesting that the model could adequately fit the experimental data.[23] Table 2. The independent variables and their levels used for Furthermore, the P-value and t ratio were used to Box–Behnken design. determine the significance of the coefficients of the Factor levels quadratic polynomial models. The smaller P-value and X −10 +1 i larger t ratio would indicate a more significant effect on Irradiation time (s) 25 50 75 [24] Ethanol percentage (%) 30 50 70 the corresponding variables. The results revealed Microwave power (%) 60 80 100 that the mathematical model was reliable for predicting SEPARATION SCIENCE AND TECHNOLOGY 5

Table 3. Experimental (Exp.) and predicted (Pred.) values of TPC and antioxidant activities (ABTS, DPPH and FRAP) of the alga S. vestitum obtained from Box–Behnken design. Microwave conditions Antioxidant activity TPC (mg GAE⋅g−1) ABTS (mg TE⋅g−1) DPPH (mg TE⋅g−1) FRAP (mg TE⋅g−1)

X1 X2 X3 Run (s) (%) (%) Exp. Pred. Exp. Pred. Exp. Pred. Exp. Pred. 1 25 70 80 57.04 57.17 145.19 149.10 106.05 109.27 60.05 58.46 2 25 30 80 53.40 54.85 140.13 139.41 107.32 109.46 58.94 55.82 3 25 50 100 50.12 50.07 144.89 142.70 105.88 101.46 36.48 43.41 4 25 50 60 53.03 51.50 144.14 143.14 97.32 96.38 47.28 45.06 5 50 30 60 54.65 54.73 140.73 142.45 104.48 103.28 38.11 43.45 6 50 70 60 57.71 59.10 154.55 151.62 114.95 112.67 32.26 36.07 7 50 50 80 56.57 56.71 151.05 152.74 110.98 111.13 62.44 63.42 8 50 50 80 57.79 56.71 152.78 152.74 112.99 111.13 66.92 63.42 9 50 70 100 56.18 56.10 152.67 150.95 109.78 110.98 62.48 57.14 10 50 50 80 55.78 56.71 154.38 152.74 109.43 111.13 60.90 63.42 11 50 30 100 55.45 54.05 142.11 145.02 104.95 107.23 33.02 29.21 12 75 30 80 56.78 56.64 145.86 141.59 111.59 108.37 52.78 54.37 13 75 50 100 50.81 52.33 143.50 144.50 102.23 103.17 52.43 54.65 14 75 50 60 54.55 54.60 139.98 142.17 101.57 105.99 53.11 46.18 15 75 70 80 62.19 60.74 146.66 147.38 123.81 121.68 69.17 72.29 Valid. 75 70 80 58.20 ± 1.71a 60.74 ± 3.84a 149.84 ± 2.71b 147.38 ± 8.42b 116.54 ± 3.65c 121.68 ± 9.46c 67.95 ± 2.63d 72.29 ± 15.29d In validation row (valid.), all the values are means ± standard deviations (n = 4), and the different letters between Exp. and Pred. values are significantly different (P < 0.05).

Table 4. Regression coefficients of the fitted quadratic equation for the TPC and antioxidant activities (ABTS, DPPH and FRAP). TPC ABTS DPPH FRAP Regression coefficients Regression coefficient t ratio Regression coefficient t ratio Regression coefficient t ratio Regression coefficient t ratio

β0 56.71 56.94 152.74 69.92 111.13 45.28 63.42 15.99 Linear β1 1.34 2.20 0.21 0.15 2.83 1.88 3.09 1.27 β2 1.60* 2.63 3.78* 2.83 3.28 2.18 5.14 2.12 β3 −0.92 −1.51 0.47 0.35 0.57 0.38 1.71 0.70 Interaction β12 0.44 0.51 −1.07 −0.56 3.37 1.59 3.82 1.11 β13 −0.21 −0.24 0.69 0.37 −1.98 −0.93 2.53 0.74 β23 −0.58 −0.67 −0.82 −0.43 −1.41 −0.66 8.83 2.57 Quadratic β11 −1.62 −1.80 −6.33* −3.22 −2.87 −1.30 1.34 0.37 β22 2.25 2.51 −1.94 −0.99 3.92 1.77 −4.52 −1.26 β33 −2.97* −3.31 −3.28 −1.66 −6.52* −2.95 −17.43* −4.87 R2 0.88 0.81 0.84 0.89 P-values of lack of fit 0.2 0.11 0.10 0.12 P-values of models 0.06 0.17 0.12 0.06

β0 is a constant, βi, βij and βii are the linear, interactive and quadratic coefficients of the second-order polynomial equation, respectively. *Significantly difference with P<0.05.

TPC values, following the second-order polynomial between the predicted and experimental values for formula (Eq. 2). ABTS, DPPH and FRAP, respectively (P>0.05). From the data above, it can be suggested that there ¼ : þ : þ : : þ : YTPC 56 71 1 34X1 1 60X2 0 92X3 0 44X1X2 was a reliability of these mathematical models in pre- : : : 2 0 21X1X3 0 58X2X3 1 62X1 dicting the antioxidant activities for the following sec- þ : 2 : 2 ond-order polynomial formulas (Eqs. 3, 4 and 5): 2 25X2 2 97X3 (2) YABTS ¼ 152:74 þ 0:21X1 þ 3:78X2 þ 0:47X3 Fitting of the models for the three different antioxidant 1:07X1X2 þ 0:69X1X3 0:82X2X3 (3) assays was also investigated. The results indicated that 6:33X2 1:94X2 3:28X2 R2 values for the models of ABTS, DPPH and FRAP 1 2 3 were 0.81, 0.84 and 0.89, respectively. The predicted values fitted fairly well with the experimental ones YDPPH ¼ 111:13 þ 2:83X1 þ 3:28X2 þ 0:57X3 obtained from the RSM design (Table 3). The values þ 3:37X X 1:98X X 1:41X X (4) for lack of fit were 0.11, 0.10 and 0.12, respectively, 1 2 1 3 2 3 : 2 þ : 2 : 2 indicating that there was no significant difference 2 87X1 3 92X2 6 52X3 6 T. T. DANG ET AL.

YFRAP ¼ 63:42 þ 3:09X1 þ 5:14X2 þ 1:71X3 þ 3:82X1X2 TPC of the S. vestitum extract (P<0.05). Based on the regression coefficients, TPC was the most affected by þ 2:53X X þ 8:83X X þ 1:34X2 1 3 2 3 1 ethanol concentration, followed by irradiation time and : 2 : 2 4 52X2 17 43X3 power of microwave. The significant effect of the quad- (5) ratic term of microwave power on the TPC of the algal extract was also observed (P<0.05). Figure 1a shows that there was a steady increase in TPC of the extract Effects of the MAE conditions on TPC when irradiation time increased from 25 to 65 s and ethanol percentage rose between 30% and 70% at the The optimal levels of the independent variables for the constant power of 80%. The value of TPC decreased a TPC were visualised by the 3D surface and 2D contour. little as irradiation time was over 65 s. In Figure 1b, The relationship between the independent variables TPC increased gradually when an increase in irradia- and the responses was shown by response surface tion time to 60 s and a medium level of irradiation plots, while the contour plot indicated the shape of a power (80%) were applied at the constant ethanol per- response surface. The effect of the irradiation time, centage of 50%. In addition, at the constant time of 50 ethanol concentration and irradiation power on TPC s, the high ethanol percentage and medium power is presented in Table 4 by the coefficients of quadratic resulted in the high yield for TPC of the extract models. As for the results, the independent variable (Figure 1c). From the model, it was predicted that the (ethanol percentage) had a significant impact on the

Figure 1. The 3D response surface and 2D contour plots (a–c) of TPC (mg GAE⋅g−1)ofS. vestitum extract mutually affected by the irradiation time (s), ethanol concentration (%) and microwave power (% or W). SEPARATION SCIENCE AND TECHNOLOGY 7

− maximum value of TPC (61.13 mg GAE⋅g 1) could be Effects of the MAE conditions on antioxidant obtained with the following conditions: irradiation time capacity of 65 s, ethanol concentration of 70% and microwave Total antioxidant capacity (ABTS) power of 70% or 840 W. For ABTS, all three parameters of the irradiation time, The effects of the irradiation time, solvent concentration ethanol concentration and microwave power affected and microwave power on the phenolics of algae and plants ABTS values of the S. vestitum extract (Figure 2a–c). were outlined by several studies. In case of the percentage of The effect of the independent variables can be shown in solvent, He et al.[25] outlined that the maximum TPC was the following order: ethanol concentration > power of obtained from the alga Saaccharina japonica with an etha- microwave > irradiation time. The statistical results nol concentration of 55%. However, in our work, the high indicated that the linear term of ethanol percentage percentage of ethanol (70%) resulted in the high yield of the and quadratic term of irradiation time significantly TPC. The difference could be due to the different constitu- affected ABTS levels (P<0.05, Table 4). It was shown ents and ratios of phenolic compounds in the extracts. It in Figure 2a that the ABTS values of S. vestitum rapidly can be seen that almost all phenolics are the polar molecules grew up when the concentration of ethanol increased that are easily dissolved by polar solvents. The proportion between 30% and 70% and irradiation time from 25 s to of ethanol in the mixture helps manipulate the polarity of 50 s at the constant power of 80%. However, the anti- the mixture that is suitable with the polarity of components oxidant activity was found to be decreased as the time for the extraction.[26] It could be that phenolic compounds was over 50 s. For Figure 2b, the ABTS activity reached from our study have the medium polarity; therefore, they at a peak as the middle levels of irradiation time and required high ethanol percentage to dissolve. In addition, as microwave power were applied for extraction at the ethanol percentage increased, more phenolic compounds constant ethanol concentration of 50%. In addition, at were extracted due to the viscosity of the media reduced by the constant irradiation time of 50 s, the high ethanol the precipitation of some polysaccharides such as lami- percentage and the medium power of microwave narin, alginate or fucoidan. With regard to irradiation resulted in the high ABTS activity (Figure 2c). time and power, it was supported by Fayad et al.,[27] who Therefore, it can be seen from the model that the indicated that the optimal condition for extracting the − highest predicted value of ABTS (154.57 mg TE⋅g 1) compounds of the alga Padina pavonica was 2 min with of the S. vestitum extract could be obtained at the microwave power of 1000 W. It is clear that high power optimal parameters (irradiation time of 50 s, ethanol increased rapidly temperature of the extraction media due percentage of 70% and power of 80%). to more microwave energy being converted into heat.[21] The high temperature helped improve the solubility of compounds, diffusion rate and mass transfer between the The DPPH free radical scavenging capacity (DPPH) solvent and material matrix. On the other hand, He et al.[25] Statistical results showed that only the quadratic term outlined that the time of 25 min was required for the of microwave power was significantly affected by extraction process of the algae Saccharina japonica with DPPH free radical scavenging capacity of the extract the irradiation power of 400 W. It was a long time com- (P<0.05; Table 4). The order of the influence of the pared to our findings with only 75 s and power of 960 W. It parameters on the values of DPPH was ethanol per- is clear that a low microwave power resulted in a long time centage > irradiation time > power. Figure 3a illus- for extraction and decrease in the yield of TPC by hydro- trates that the DPPH value of the S. vestitum extract lysation and oxidation of some polyphenols at a high rose gradually when irradiation time increased in the temperature.[28] From the findings, it is suggested that range of 25 and 75 s and ethanol concentration using a high power with a short time is better than a low increased between 30% and 70% min at the power power with a long time. However, as irradiation power was of 80%. In Figure 3b, at an ethanol level of 70%, the over 80% with a long time treatment, a degradation of high DPPH value was shown at the time of 75 s and thermal sensitivity compounds could happen in algal the medium power of microwave. With the irradia- extracts.[29] From the model, it was found that the highest tion time of 50 s, the moderate power and high – yield of TPC (61.13 mg GAE⋅g 1) could be obtained at the concentration of ethanol led to high value of DPPH optimal conditions of the irradiation time of 65 s, ethanol of the extract (Figure 3c). From the results, the opti- percentage of 70% and microwave power of 70%. mal parameters for DPPH activity were irradiation time of 75 s, ethanol concentration of 70% and microwave power of 80%. The maximal value for − DPPH activity (121.68 mg TE⋅g 1) could be obtained at the predicted conditions above. 8 T. T. DANG ET AL.

Figure 2. The 3D response surface and 2D contour plots (a–c) of ABTS (mg TE⋅g−1)ofS. vestitum extract mutually affected by the irradiation time (s), ethanol concentration (%) and microwave power (% or W).

Ferric-reducing antioxidant power (FRAP) ranged from 60% to 80%, but FRAP decreased quickly The results from Figure 4a–c and Table 4 indicated as power was over 90% (1080 W) at ethanol percentage changes in FRAP activity of the extract under the of 50% (Figure 4b). It was shown in Figure 4c that at different irradiation conditions. As a result, the FRAP the irradiation time of 50 s the high value of FRAP was value was significantly affected by the quadratic term of obtained when percentage of ethanol at 60% and the microwave power (P<0.05). The trend of FRAP activ- moderate microwave power was applied. From the ity was similar to TPC and DPPH with the most effect model, it was shown that the maximum value of − by the ethanol concentration, followed by the irradia- FRAP (74.46 mg TE⋅g 1) could be obtained with the tion time and power of microwave. The quick increase conditions of the irradiation time (75 s), the percentage in the values of FRAP was observed as the irradiation of ethanol (70%) and the power (90% or 1080 W). It time increased (range of 25 and 75 s) and ethanol can be noted that a positive relationship between phe- percentage ranged between 30% and 70% at the mod- nolic compounds and antioxidant capacity of the algal – erate power of 80% (Figure 4a). The antioxidant activity extracts has been highlighted by several studies.[30 32] It of the extract grew up slightly as the irradiation time means that the factors made the increase in the yield of increased and rose rapidly when the irradiation power TPC also increased the antioxidant activities of the SEPARATION SCIENCE AND TECHNOLOGY 9

Figure 3. The 3D response surface and 2D contour plots (a–c) of DPPH (mg TE⋅g−1)ofS. vestitum extract mutually affected by the irradiation time (X1, s), ethanol concentration (X2, %) and microwave power (X3, % or W). extract. From the findings, as the irradiation time and release of the components from the cell rupture and ethanol percentage increased, more active compounds the relationship between the irradiation time and power were extracted that increased the antioxidant activities for the treatment of the sample (10 and 15 min at 200 of the extracts. DPPH and FRAP values were high as W; 10 min at 400 W; 7 min at 600 W; 5 min at 800 W the irradiation time and ethanol percentage were at the and 4 min at 900 W for the maximal yields). In our high levels of 75 s and 70%, respectively. However, work, the higher power of 80–90% (960–1080 W) was ABTS activity at the top with the irradiation time of applied, and shorter time (65–75 s) was required for the 50 s indicated that some phenolic compounds may be maximum of the TPC and antioxidant capacity of the vulnerable to the irradiation treatment. In addition, the extract. Ying et al.[34] also indicated that the maximum microwave power at the middle level of 70–80% (840– DPPH free radical scavenging activity of Porphyra 960 W) was suitable to obtain high values of three yezoensis extract was obtained under the following antioxidant assays tested. experimental conditions: the microwave power of 500 It was in line with Pérez et al.,[33] who showed that W and the irradiation time of 6 min. On the other an increase in the microwave power led to a high ABTS hand, it was different in comparison to the result of the brown alga Sargassum muticum due to the shown by Lin et al. [35] that the low percentage of 10 T. T. DANG ET AL.

Figure 4. The 3D response surface and 2D contour plots (a–c) of FRAP (mg TE⋅g−1)ofS. vestitum extract mutually affected by the irradiation time (X1, s), ethanol concentration (X2, %) and microwave power (X3, % or W). ethanol (10%) resulted in the high DPPH value of the FRAP). As per the discussion above and outlined in extract Monostroma nitidum. It could be that the dif- Figure 5, the increase in the irradiation time and etha- ferent species led to differences in the constituents and nol percentage with the medium microwave power led the proportions of the compounds in extract. The dif- to the increase in TPC and antioxidant activities of the ferent ethanol percentage in the mixture with water extract. It is illustrated in Figure 5 that the theoretical changed the polarity of the mixture that was suitable maximum values of TPC, DPPH and FRAP could be for extraction of phenolic compounds. At a high etha- obtained by combining the irradiation time, ethanol nol percentage used in our work, more medium-polar percentage and microwave power. Based on the predic- phenolic were extracted, while more polysaccharides tion of the model, the highest TPC, DPPH and FRAP − − were removed due to the precipitation of the polysac- obtained were 60.74 mg GAE⋅g 1, 121.68 mg TE⋅g 1 − charides. Thus, using the high ethanol concentration and 72.29 mg TE⋅g 1, respectively, with the optimal (70%) made the high DPPH activity of the extract. conditions (the irradiation time 75 s, ethanol percen- tage of 70% and microwave power of 80%). The ABTS − value was 147.38 mg TE⋅g 1 with these parameters Optimisation and validation of MAE conditions (gained 95.33% of the predicted maximum value of The study aimed to determine the optimal conditions ABTS activity with the conditions that were similar to for TPC and antioxidant activities of the alga S. vesti- the parameters above, except for the irradiation time of tum using a JMP 13 software. Through the process, the 50 s). Therefore, these parameters were chosen as the irradiation time, ethanol concentration and microwave optimal conditions for extracting the alga S. vestitum. power were estimated to obtain the maximum values In addition, the experiments were performed under for TPC and antioxidant activities (ABTS, DPPH and these optimal conditions to validate the adequacy of SEPARATION SCIENCE AND TECHNOLOGY 11

Figure 5. The predicted profilers of TPC and antioxidant activities of S. vestitum extract at the optimal conditions of the irradiation time (X1, s), ethanol concentration (X2, %) and microwave power (X3, % or W). Solid lines indicate predicted mean values of TPC and antioxidant activities. Red dashed lines show the values at each condition, while blue lines indicate the 95% confidence intervals. The predicted profilers were predicted by response surface methodology using JMP software (version 13). the predicted models. The statistical results illustrated percentage of 70% and the ultrasonic power of 150 W). that there was no significant difference between pre- The conditions for the conventional protocol were also dicted and measured responses of TPC and antioxidant applied with the time of 12 h, the temperature of 30°C activities (ABTS, DPPH and FRAP) (P>0.05; Table 3). and ethanol percentage of 70%. It is evident that the measured values of the responses The results exhibited that the MAE was effective to were found to be well fitted to the predicted ones by the obtain significantly higher levels of TPC and antiox- regression model. Therefore, these conditions were sug- idant capacity of the extract when compared to the gested to extract the high yield of TPC and antioxidant conventional and ultrasonic methods (P<0.05; activities of S. vestitum for further isolation and utilisa- Table 5). The value of TPC achieved using the micro- tion. The results also confirmed appropriateness of the wave irradiation was 144.38% and 120.12% higher models used for optimising the extraction conditions than the ones by the conventional and ultrasonic using MAE technique. extraction, respectively. In terms of antioxidant capa- city, the values for ABTS, DPPH and FRAP using the microwave were also higher (133.98%, 162.58% and Comparison of different extraction techniques 146.95%, respectively) compared with those of the The comparison between three methods of convention, conventional method. Both DPPH and FRAP activ- UAE and MAE was estimated to assess recovery effi- ities of the microwave extracts were significantly cacy of the TPC and antioxidant activities of the S. higher than of the ultrasonic treatments, while no vestitum extract. The optimal MAE conditions were significant difference was found in ABTS activity of used to compare with UAE procedure (the ultrasonic these methods. It can be noted that the time applied time of 60 min, the temperature of 30°C, the ethanol for the microwave extraction was only 75 s, whereas

Table 5. Comparison of the efficacy for TPC and antioxidant capacity of the extracts using the MAE, UAE and conventional method. Extraction methods Conventional extraction UAE MAE TPC (mg GAE⋅g−1) 40.31 ± 2.14c 48.45 ± 0.20b 58.20 ± 1.71a ABTS (mg TE⋅g−1) 111.83 ± 3.27b 147.19 ± 0.45a 149.84 ± 2.71a DPPH (mg TE⋅g−1) 71.68 ± 1.63c 86.07 ± 2.07b 116.54 ± 3.65a FRAP (mg TE⋅g−1) 46.24 ± 2.20c 60.10 ± 1.48b 67.95 ± 2.63a All the values are means ± standard deviations (n = 3), and the different letters in the same row are significantly different (P<0.05). 12 T. T. DANG ET AL. it was required for the ultrasound and conventional [3] El Gamal, A.A.;. (2010) Biological importance of mar- conditions to be 1 and 12 h, respectively. The find- ine algae. Saudi Pharmaceutical Journal, 18 (1): 1–25. ings confirmed the advantages of the microwave [4] Mohamed, S.; Hashim, S.N.; Rahman, H.A. (2012) Seaweeds: a sustainable functional food for complementary method in terms of the efficacy and quality of the and alternative therapy. Trends in Food Science & extracts for extracting biologically active compounds Technology,23(2):83–96. of the alga. [5] Cox, S.; Abu-Ghannam, N.; Gupta, S. (2010) An assess- ment of the antioxidant and antimicrobial activity of six species of edible Irish seaweeds. International Food Conclusions Research Journal, 17: 205–220. [6] Wang, T.; Jonsdottir, R.; Ólafsdóttir, G. (2009) Total The results indicated that the quadratic polynomial phenolic compounds, radical scavenging and metal models were sufficient to describe and predict the chelation of extracts from Icelandic seaweeds. Food responses of the TPC and antioxidant activities Chemistry, 116 (1): 240–248. (ABTS, DPPH and FRAP) in the optimisation pro- [7] Wang, L.; Weller, C.L. (2006) Recent advances in extraction of nutraceuticals from plants. Trends in cess. The optimal microwave conditions for extrac- Food Science & Technology, 17 (6): 300–312. tion of phenolics were as follows: the irradiation time [8] Azmir, J.; Zaidul, I.; Rahman, M.; Sharif, K.; Mohamed, of 75 s, ethanol percentage of 70% and microwave A.; Sahena, F.; Omar, A. (2013) Techniques for extrac- power of 960 W. In comparison to the conventional tion of bioactive compounds from plant materials: a – and ultrasonic method, the advantages of MAE for review. Journal of Food Engineering, 117 (4): 426 436. [9] Polshettiwar, V.; Varma, R.S. (2008) Aqueous micro- extraction of the active components of the algae were wave chemistry: a clean and green synthetic tool for short time and high microwave power with the short rapid drug discovery. Chemical Society Reviews, 37 (8): time for the treatment not affecting much the anti- 1546–1557. oxidant activities of the extract. The extraction con- [10] Sticher, O.;. (2008) Natural product isolation. Natural ditions were potentially applied for extraction, Product Reports, 25 (3): 517–554. isolation and purification of phenolics of the alga S. [11] Zhang, H.F.; Yang, X.H.; Wang, Y. (2011) Microwave assisted extraction of secondary metabolites from vestitum and for further application in the food and plants: current status and future directions. Trends in pharmaceutical industries. Food Science & Technology, 22 (12): 672–688. [12] Tatke, P.; Jaiswal, Y. (2011) An overview of microwave assisted extraction and its applications in herbal drug Acknowledgments research. Researcher Journal Medica Plant, 5 (1): 21–31. [13] Kala, H.K.; Mehta, R.; Sen, K.K.; Tandey, R.; Mandal, The authors kindly thank the University of Newcastle, the V. (2016) Critical analysis of research trends and issues Vietnamese Government through the Vietnam International – in microwave assisted extraction of phenolics: have we Education Development Ministry of Education and really done enough. TrAC Trends in Analytical Training and the Ministry of Agriculture and Rural Chemistry, 85: 140–152. Development for awarding a VIED-TUIT scholarship to [14] Anderson-Cook, C.M.; Borror, C.M.; Montgomery, D. ThanhTrung DANG. C. (2009) Response surface design evaluation and com- parison. Journal of Statistical Planning and Inference, 139 (2): 629–641. Declaration of interest [15] Alonso-Carrillo, N.; De Los Angeles Aguilar- The authors declare no conflict of interest. Santamaria, M.; Vernon-Carter, E.J.; Jimenez- Alvarado, R.; Cruz-Sosa, F.; Roman-Guerrero, A. (2017) Extraction of phenolic compounds from Funding Satureja macrostema using microwave-ultrasound assisted and reflux methods and evaluation of their The authors would like to acknowledge the following funding antioxidant activity and cytotoxicity. Industrial Crops support: Ramaciotti Foundation [ES2012/0104]. and Products, 103: 213–221. [16] Hossain, M.B.; Brunton, N.P.; Patras, A.; Tiwari, B.; O’Donnell, C.; Martin-Diana, A.B.; Barry-Ryan, C. References (2012) Optimization of ultrasound assisted extraction of antioxidant compounds from marjoram (Origanum [1] Gupta, S.; Abu-Ghannam, N. (2011) Bioactive potential majorana L.) using response surface methodology. and possible health effects of edible brown seaweeds. Ultrasonics Sonochemistry, 19 (3): 582–590. Trends in Food Science & Technology, 22 (6): 315–326. [17] Skerget, M.; Kotnik, P.; Hadolin, M.; Hraš, A.R.; [2] Balboa, E.M.; Conde, E.; Moure, A.; Falque, E.; Simonič, M.; Knez, Z. (2005) Phenols, proanthocyani- Dominguez, H. (2013) In vitro antioxidant properties dins, flavones and flavonols in some plant materials of crude extracts and compounds from brown algae. and their antioxidant activities. Food Chemistry, 89 (2): Food Chemistry, 138 (2): 1764–1785. 191–198. SEPARATION SCIENCE AND TECHNOLOGY 13

[18] Thaipong, K.; Boonprakob, U.; Crosby, K.; Cisneros- conventional solvent extraction. Chemical Engineering Zevallos, L.; Hawkins Byrne, D. (2006) Comparison and Processing: Process Intensification116: 29–39. of ABTS, DPPH, FRAP, and ORAC assays for esti- [27] Fayad, S.; Nehme, R.; Tannoury, M.; Lesellier, E.; mating antioxidantactivity from guava fruit extracts. Pichon, C.; Morin, P. (2017) Macroalga Padina pavo- Journal of Food Composition and Analysis,19(6): nica water extracts obtained by pressurized liquid 669–675. extraction and microwave assisted extraction inhibit [19] Brand-Williams, W.; Cuvelier, M.; Berset, C. (1995) Use hyaluronidase activity as shown by capillary electro- of a free radical method to evaluate antioxidant activity. phoresis. Journal of Chromatography A, 1497: 19–27. LWT-Food Science and Technology, 28 (1): 25–30. [28] Naczk, M.; Shahidi, F. (2004) Extraction and analysis of [20] Benzie, I.; Strain, J. (1999) Ferric reducing/antioxidant phenolics in food. Journal of Chromatography A, 1054 power assay: direct measure of total antioxidant activ- (1): 95–111. ity of biological fluids and modified version for simul- [29] Liazid, A.; Palma, M.; Brigui, J.; Barroso, C.G. (2007) taneous measurement of total antioxidant power and Investigation on phenolic compounds stability during ascorbic acid concentration. Methods in Enzymology, microwave-assisted extraction. Journal of 299: 15–27. Chromatography A, 1140 (1): 29–34. [21] Yan, M.M.; Liu, W.; Fu, Y.J.; Zu, Y.G.; Chen, C.Y.; Luo, [30] Le Lann, K.; Jegou, C.; Stiger-Pouvreau, V. (2008) M. (2010) Optimisation of the microwave-assisted Effect of different conditioning treatments on total extraction process for four main astragalosides in phenolic content and antioxidant activities in two Radix Astragali. Food Chemistry, 119 (4): 1663–1670. Sargassacean species: Comparison of the frondose [22] Guo, Z.; Jin, Q.; Fan, G.; Duan, Y.; Qin, C.; Wen, M. Sargassum muticum (Yendo) Fensholt and the cylind- (2001) Microwave-assisted extraction of effective con- rical Bifurcaria bifurcata R. Ross. Phycological Research, stituents from a Chinese herbal medicine. Radix 56 (4): 238–245. Puerariae. Analytica Chimica Acta, 436 (1): 41–47. [31] Jimenez-Escrig, A.; Jimenez-Jimenez, I.; Pulido, R.; [23] Wu, Y.; Wang, X.; Fan, E. (2012) Optimisation of Saura-Calixto, F. (2001) Antioxidant activity of fresh ultrasound-assisted extraction of puerarin and total and processed edible seaweeds. Journal of the Science of isoflavones from puerariae lobatae radix (Pueraria Food and Agriculture, 81 (5): 530–534. lobata (Wild.) Ohwi) with response surface methodol- [32] Dang, T.T.; Vuong, Q.V.; Schreider, M.J.; Bowyer, M. ogy. Phytochemical Analysis, 23 (5): 513–519. C.; Altena, I.A.V.; Scarlett, C.J. (2016) The effects of [24] Kha, T.C.; Nguyen, M.H.; Phan, D.T.; Roach, P.D.; drying on physico-chemical properties and antioxidant Stathopoulos, C.E. (2013) Optimisation of microwave- capacity of the brown alga (Hormosira banksii (Turner) assisted extraction of Gac oil at different hydraulic Decaisne). Journal of Food Processing and Preservation, pressure, microwave and steaming conditions. Doi: 10.1111/jfpp.13025. International Journal of Food Science & Technology, [33] Perez, L.; Conde, E.; Dominguez, H. (2014) Microwave 48 (7): 1436–1444. hydrodiffusion and gravity processing of Sargassum [25] He, Z.; Chen, Y.; Chen, Y.; Liu, H.; Yuan, G.; Fan, Y.; muticum. Process Biochemistry, 49 (6): 981–988. Chen, K. (2013) Optimization of the microwave- [34] Ying, W.; Lei, G. (2012) Optimization of microwave- assisted extraction of phlorotannins from Saccharina assisted extraction conditions of DPPH radical scaven- japonica Aresch and evaluation of the inhibitory effects ging components from Porphyra yezoensis by response of phlorotannin-containing extracts on HepG2 cancer surface methodology. Paper presented at the cells. Chinese Journal of Oceanology and Limnology, 31: Information Technology in Medicine and Education 1045–1054. (ITME), 2012 International Symposium. DOI: 10.1109/ [26] Galan, A.M.; Calinescu, I.; Trifan, A.; Winkworth-Smith, ITiME.2012.6291480. C.; Calvo-Carrascal, M.; Dodds, C.; Binner, E. (2017) New [35] Lin, Y.P.; Huang, S.L.; Wu, S.C. (2013) Effects of insights into the role of selective and volumetric heating microwave-assisted extraction on the free radical during microwave extraction: investigation of the extrac- scavenging and ferrous chelating abilities of Porphyra tion of polyphenolic compounds from sea buckthorn dentate extract. Marine Science and Technology, 38 (4): leaves using microwave-assisted extraction and 317–328. Paper V:

“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 BotanicaMarina.

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1 2 3 be more important in the activity of the extract. However, Cox et al. (2010) outlined that 4 5 flavonoids and tannins may be the principal constituents responsible for antioxidant activity 6 7 of algal extracts, while Cho et al. (2010) demonstrated Àavonoids, tannins and phenolic acid 8 9 rather than total phenolic content accounted for antioxidant activity of the extracts. Lim et al. 10 11 (2002) also indicated that no correlation between TPC and antioxidant activity was observed 12 13 14 from the brown alga Sargassum siliquastrum. Therefore, the species-specific differences, 15 16 affected both the composition and their ratios, led to difference in antioxidant activity of algal 17 18 extracts. In our studies, total phenolics, flavonoids and tannins all contributed to antioxidant 19 20 activities (ABTS, DPPH and FRAP), while phenolics in both EA and BuOH fractions were 21 22 major contributors to these activities of the H. banksii extract. 23 24 Conclusions 25 26 27 This study indicated that the crude extract H. banksii was a rich source of phenolics. The EA 28 29 fraction possessed the highest levels of TPC, tannins and antioxidant activities comparable to 30 31 positive controls (BHT, ascorbic acid and Į-tocopherol). The difference in the ratio of the 32 33 components and antioxidant activities among the fractions indicated the effectiveness of 34 35 solvent-partition technique for isolation of the active compounds from the Hormosira banksii 36 37 extract. The phenolics in the EA and BuOH fraction were considered as the main antioxidants 38 39 40 continuously isolated and identified structures and further utilised in the food and 41 42 pharmaceutical industries. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/botmar Botanica Marina Page 16 of 29

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“Fucoxanthin content, isolation and cytotoxic activity against pancreatic cancer from brown alga Hormosira banksii (Turner) Decaisne”. Submitted to Journal of Biotechnology.

Elsevier Editorial System(tm) for Journal of Biotechnology Manuscript Draft

Manuscript Number:

Title: Fucoxanthin content, isolation and cytotoxic activity against pancreatic cancer from brown alga Hormosira banksii (Turner) Decaisne

Article Type: Research Paper

Section/Category: Medical Biotechnology

Keywords: Fucoxanthin content; Hormosira banksii; Brown algae; HPLC analysis; Pancreatic cancer.

Corresponding Author: Mr. Thanh T Dang,

Corresponding Author's Institution: School of Environmental and Life Sciences

First Author: Thanh T Dang

Order of Authors: Thanh T Dang; Deep J Bhuyan; Danielle R Bond; Michael C Bowyer; Ian A Van Altena; Christopher J Scarlett

Abstract: Fucoxanthin content has recently gained increased attention due to its health benefits. This study aimed to isolate fucoxanthin from the brown alga Hormosira banksii and fucoxanthin was investigated for cytotoxic activity against pancreatic cell lines. The HPLC analysis showed that fucoxanthin content in the alga was determined to be 0.58 mg Fx.g-1 alga, while its purity achieved at 92.3% via column chromatography. This pigment showed high anti-proliferative activity on both primary (MiaPaCa2) and secondary (BxPC3, CFPAC1) pancreatic cancer cell lines at concentrations of 100-200 μg.mL-1. The fucoxanthin was successfully isolated from this specie and exhibited high activity against pancreatic cancer. It provided rationale for future clinical use of fucoxanthin for the treatment of pancreatic cancer and investigations into the mechanism of action of this compound on apoptosis and cell cycle.

Suggested Reviewers: Jun Lu School of Applied Sciences, Faculty of Health and Environment Sciences, School of Applied Sciences, Faculty of Health and Environment Sciences, Auckland University of Technology, Auckland 1142, New Zealand [email protected] He is an expert in biomedical technology

Fatimah Md. Yusoff Department of Aquaculture, Faculty of Agriculture, Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia [email protected] He is an expert in agriculture

You-Jin Jeon Laboratory of Veterinary Molecular Pathology and Therapeutics, Laboratory of Veterinary Molecular Pathology and Therapeutics, Tokyo University of Agricultural and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan [email protected] He is an expert in Molecular Pathology and Therapeutics

Xia-min Hu Department of pharmacology, Department of pharmacology, Medical college of Wuhan University of Science and Technology, Wuhan 430080, China [email protected] He is an expert in pharmaceutical field Cover Letter

Saturday 28th April, 2018

Prof. Christoph W. Sensen Editor-in-chief Journal of Biotechnology

Dear Prof. Christoph W. Sensen,

On behalf of my co-authors, I would like to submit our original manuscript entitled “Fucoxanthin content, isolation and cytotoxic activity against pancreatic cancer from brown alga Hormosira banksii (Turner) Decaisne”, which we wish to be considered for publication as an original research article in Journal of Biotechnology.

Hormosira banksii is a brown fucoid alga rich in bioactive compounds, and is widely distributed in the intertidal areas along the eastern coast of Australia. Fucoxanthin content has recently gained increased attention due to its health benefits. This study aimed to isolate and purify fucoxanthin from the brown alga Hormosira banksii and the purified fucoxanthin was investigated for cytotoxic activity against pancreatic cell lines. The HPLC analysis showed that fucoxanthin content in the alga was determined to be 0.58 mg Fx.g-1 alga, while its purity was achieved at 92.3% via column chromatography. This pigment showed high anti-proliferative activity on both primary (MiaPaCa2) and secondary (BxPC3, CFPAC1) pancreatic cancer cell lines at concentrations of 100-200 μg.mL-1. It provided rationale for future clinical use of fucoxanthin for the treatment of pancreatic cancer and investigations into the mechanism of action of this compound on apoptosis and cell cycle.

Our manuscript reports previously unpublished work and has not been submitted simultaneously, in whole or in part, to another journal. The manuscript has been seen and approved by all authors and have agreed to submit this manuscript to Journal of Biotechnology in its present form. We hope that you and the reviewers share our enthusiasm for these data, and we look forward to receiving your comments in due course.

With my best regards, A/Prof. Christopher Scarlett, PhD

Deputy Head of School (Ourimbah) Head Discipline of Applied Science, School of Environmental and Life Sciences. Head, Pancreatic Cancer Research, University of Newcastle. 10 Chittaway Rd, Ourimbah, NSW 2258, Australia. Tel.: +61 2 4348 4680; Fax: +61 243484145; E-mail: [email protected] *Highlights (for review)

Highlights

- Fucoxanthin content from Hormosira banksii was successfully isolated via a chromatography column with the purity of 92.3%.

- Fucoxanthin content was confirmed and estimated of 0.58 mg Fx.g-1 dry alga by HPLC analysis.

- Fucoxanthin showed strong anticancer activity against pancreatic cancer cell lines. 1 Fucoxanthin content, isolation and cytotoxic activity against pancreatic cancer 2 from brown alga Hormosira banksii (Turner) Decaisne

3 Thanh T. Dangab, Deep J. Bhuyana, Danielle R. Bondac, Michael C. Bowyera, Ian A. Van 4 Altenaa & Christopher J. Scarletta*

5 a School of Environmental and Life Sciences, Faculty of Science, University of Newcastle, Ourimbah, New South Wales, 6 Australia 7 b Department of Seafood Processing Technology, Faculty of Food Technology, Nha Trang University, Nha Trang, Khanh 8 Hoa, Vietnam 9 c School of Biomedical Science & Pharmacy, Faculty of Health and Medicine, University of Newcastle, Ourimbah, New 10 South Wales, Australia

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16 Running Title: Fucoxanthin content and anticancer activity of H. banksii.

17

18 *Corresponding author:

19 A/Prof. Christopher J. Scarlett, PhD

20 Deputy Head of School (Ourimbah). 21 Head Discipline of Applied Science, 22 School of Environmental and Life Sciences. 23 Head, Pancreatic Cancer Research, 24 University of Newcastle. 25 10 Chittaway Rd, Ourimbah, NSW 2258, Australia. 26 27 Ph: +61 2 4348 4680 Fax: +61 2 4348 4145 28 Email: [email protected]

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1 1 Abstract

2 Fucoxanthin content has recently gained increased attention due to its health benefits. This

3 study aimed to isolate fucoxanthin from the brown alga Hormosira banksii and fucoxanthin

4 was investigated for cytotoxic activity against pancreatic cell lines. The HPLC analysis

5 showed that fucoxanthin content in the alga was determined to be 0.58 mg Fx.g-1 alga, while

6 its purity achieved at 92.3% via column chromatography. This pigment showed high anti-

7 proliferative activity on both primary (MiaPaCa2) and secondary (BxPC3, CFPAC1)

8 pancreatic cancer cell lines at concentrations of 100-200 μg.mL-1. The fucoxanthin was

9 successfully isolated from this specie and exhibited high activity against pancreatic cancer. It

10 provided rationale for future clinical use of fucoxanthin for the treatment of pancreatic cancer

11 and investigations into the mechanism of action of this compound on apoptosis and cell

12 cycle.

13

14

15 Keyword: Fucoxanthin content; Hormosira banksii; Brown algae; HPLC analysis;

16 Pancreatic cancer.

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2 1 1. Introduction

2 Marine algae are renewable and sustainable resources that contain biologically active

3 metabolites, and that could be potentially exploited for the development of new drugs and

4 healthy foods (Gupta and Abu-Ghannam, 2011). The natural components of algal extracts

5 such as phenolic compounds, fucoxanthin, sulfated polysaccharides, terpenoids and other

6 secondary metabolites were found to have a broad spectrum of biological activities (Balboa et

7 al., 2013; Mohamed et al., 2012). It has been also demonstrated that these compounds are

8 linked to the treatment of some chronic diseases, especially several types of cancers

9 (Schumacher et al., 2011; Sithranga Boopathy and Kathiresan, 2010).

10 Brown algae possess not only phenolics (phlorotannins) and polysaccharides but also

11 fucoxanthin (carotenoid) that has recently gained attention due to its potent applications in

12 the food and pharmaceutical industries (Peng et al., 2011; Wijesinghe and Jeon, 2012).

13 Fucoxanthin is one of the most abundant carotenoids of brown algae and estimated to be

14 around 10% of total carotenoids found in nature (Rajauria et al., 2017). The structure of

15 fucoxanthin includes a usual allenic bond and 5, 6-monoepoxide in its molecule and the type

16 of this compound found in brown algae was almost all trans-fucoxanthin (Figure 2) (Jaswir et

17 al., 2013; Nakazawa et al., 2009). Fucoxanthin content from the algal extracts was isolated

18 and purified through several steps such as partition to create the fucoxanthin-enriched

19 fractions (Kim et al., 2011), using preparative thin layer chromatography (TLC) to directly

20 isolate fucoxanthin from the extract (Rajauria et al., 2017), the pigments commonly eluted

21 from the fractions or crude extracts through silica column chromatography (Imbs et al., 2013;

22 Mori et al., 2004), or isolation of the pigments with HPLC system (fraction collectors). The

23 health benefits of fucoxanthin and its efficacy against several cancer cell lines have been

24 previously studied (D’Orazio et al., 2012; Imbs et al., 2013; Wang et al., 2014).

3 1 Pancreatic cancer is one the leading causes of cancer death in Western countries due to the

2 late onset of symptoms and diagnosis at an advanced stage. Pancreatic ductal

3 adenocarcinoma (PDAC) is the most common pancreatic cancer type (95%) (Behrens et al.,

4 2017; Scarlett et al., 2011), and currently, therapeutic options applicable to PDAC are still

5 limited to surgery at the early stage, while chemotherapy and radiotherapy are applied for

6 almost all PDAC patients (Raviv et al., 2017). On the other hand, chemotherapeutics using

7 synthetic drugs such as gemcitabine (2-2-difluorodeoxycytidine) or the combination of 5-

8 fluorouracil, leucovorin, oxaliplatin and irinotecan (FOLFIRINOX) treatment were found to

9 not be effective due to their significant toxicity and emerging drug resistance (Behrens et al.,

10 2017; Petrelli et al., 2017). With limited therapeutic options for pancreatic cancer, it is

11 necessary to screen the anticancer activity of active compounds found in marine plants.

12 Fucoxanthin within the algal extracts may be potentially useful for the treatment of pancreatic

13 cancer.

14 Based on our best knowledge, fucoxanthin content of brown alga Hormosira banksii

15 (collected from the eastern coast of NSW, Australia) has yet to be investigated. The aims of

16 our work were to isolate fucoxanthin from this specie, with the anticancer activity of

17 fucoxanthin then evaluated using several pancreatic cancer cell lines for potential

18 applications in pharmaceutical field.

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25

4 1 2. Materials and methods

2 2.1 Reagents

3 All solvents and chemicals such as n-hexane (95.0%), acetone (99.5%), methanol (99.8%),

4 ethanol (99.5%) and acetonitrile (99.8%) used in the study were analytical grade (Sigma-

5 Aldrich, Australia). A silica gel plate (20x20 cm, Kieselgel 60F254, 0.25 mm); Kieselgel

6 60GF254 powder for chromatography column (Merck KGaA, Darmstadt, Germany). The

7 standard fucoxanthin (95% purity) for this work was purchased from Sigma-Aldrich, Castle

8 Hill, NSW 2154, Australia.

9 2.2 Materials and sample preparation

10 The brown alga H. banksii was collected in March, 2016 from a rocky shore at Bateau Bay,

11 NSW, Australia (Latitude of 33°22'55.2"S; longitude of 151°29'6"E). The sample was

12 washed with seawater, and then washed thoroughly with freshwater and freeze dried for 48 h

13 using a freeze dryer (Thomas Australia Pvt. Ltd., Seven Hills, NSW, Australia). The dried

14 sample was pulverized, sieved ” 600 μm particle size) and stored at – 20 °C for further

15 analysis. The extraction process was performed as previously described by Fung et al. (2013)

16 with slight modifications. Briefly, the dried alga (10g) was extracted with pure ethanol (300

17 mL) and stirred using a magnetic bar for 8 h at room temperature. The process was repeated

18 (n=3) till the samples became colourless. The combined extracts were filtered, concentrated

19 using a rotary evaporator, and freeze dried to obtain the crude extracts.

20 2.3 Isolation of fucoxanthin

21 The process for isolation of fucoxanthin from the extract was conducted as described by

22 Jaswir et al. (2011) with slight modifications. Fucoxanthin was separated through a

23 chromatography column using silica gel (Kieselgel 60GF254). A clean glass column (3.5x32

24 cm) initially was inserted with cotton wool at the bottom, and silica gel (30g) dissolved with

25 the n-hexane was poured immediately into the column to avoid breakage or bubbles. The

5 1 column was kept one day for proper binding of silica. The extract dissolved in methanol was

2 loaded in the column. Pure n-hexane was initially used to elute non-polar content such as

3 chlorophyll, other carotenoids than fucoxanthin. The next elution was conducted with a

4 mixture of hexane:acetone (7:3 v/v) to obtain fucoxanthin. Finally, acetone was used to elute

5 residual fucoxanthin. The fractions of acetone and the mixture of hexane:acetone were

6 collected and concentrated using a rotary evaporator. The experiment was conducted in the

7 dark room to avoid the possibility of oxidation/degradation of fucoxanthin by light.

8 2.4 Thin layer chromatography (TLC) and HPLC analysis

9 TLC analysis for the extract and fucoxanthin was performed on a silica gel plate (20x20 cm,

10 Kieselgel 60F254, 0.25 mm). The mixture of hexane and acetone (7/3; v/v) was used as a

11 mobile phase.

12 Fucoxanthin content was determined using high performance liquid chromatography (HPLC)

13 (Terasaki et al., 2009). The HPLC system consisted of a LC-20AT pump system (Shimadzu)

14 and a UV–Vis SPD-20A (Shimadzu) absorbance detector. Fucoxanthin was separated on a

15 Luna 5 μm C18 column (250 x 4.6 mm) (Phenomenex, Australia). The mobile phase used

16 was a mixture of methanol/acetonitrile (7/3 v/v) at a ÀRZ rate of 1 ml/min and the sample

17 injection volume was 50 μl. The detection wavelength for fucoxanthin was set at 450 nm.

18 2.5 Cell culture

19 Human pancreatic cancer cells (two primary pancreatic cancer cell lines: MiaPaCa2, BxPC3

20 and one secondary pancreatic cancer cell line: CFPAC1) and human pancreatic ductal

21 epithelial cells (HPDE) were cultured at 37 °C, 5% CO2. Keratinocyte Serum-free Media (K-

22 SFM) with human Recombinant Epidermal Growth Factor (EGF) and Bovine Pituitary

23 Extract (BPE) (10%) was used for HPDE. Dulbecco’s Modified Eagle’s Medium (DMEM)

24 supplemented with 10% foetal bovine serum (FBS), 2.5% horse serum and L-glutamine (100

25 μg.mL-1) was used to culture MiaPaCa2 cells, while RPMI media with 10% FBS and L-

6 1 glutamine (100 μg.mL-1) was applied for BxPC3 and CFPAC1 cells were cultured in IMDM

2 media supplemented with 10% FBS and L-glutamine (100 μg.mL-1).

3 2.6 Cytotoxic activity of fucoxanthin

4 Cytotoxic activity of fucoxanthin was determined using the Dojindo Cell Counting Kit-8

5 (CCK-8: Dojindo Molecular Technologies, Inc., Rockville, MD, USA) assay. Pancreatic cells

6 were seeded into a 96-well plate at 3x103 cells per well (200 μL) for MiaCaPa2 and 7x103

7 cells for HPDE, BxPC3 and CFPAC1, and incubated at 37 °C with 5% CO2 for 24 h. The

8 cells were then treated with various concentrations (25-200 μg.mL-1) of fucoxanthin,

9 gemcitabine (50 n M ~13.16x10-3μg.ml-1) and 0.5% DMSO. After 72 h, 10 μLofCCK-8

10 solution was added before incubating at 37 °C with 5% CO2 for 90 min. The absorbance was

11 measured at 450 nm on a multi-well spectrophotometer (BIORAD Benchmark PlusTM), and

12 the cytotoxic activity of fucoxanthin was determined as a percentage of dead cells compared

13 to control (DMSO) (% inhibition = ((Abs of the control – Abs of the sample)/Abs of the

14 control) x 100%). Six repeats were performed for each concentration (n=6).

15 2.7 Statistical Analysis

16 A one-way ANOVA and LSD post-hoc test were employed (SPSS Statistical Software,

17 Version 16) to analyse the differences between the independent samples. Differences between

18 the mean levels of analyses were taken to be statistically significant at P<0.05. All

19 experiments were conducted at least in triplicate and the results were performed as means ±

20 standard deviations. The IC50 values (the concentration required to inhibit cell growth by

21 50%) were calculated by curve fitting the absorbance (viability) vs. log [concentration of

22 treatment] using GraphPad Prism software (version 7.03).

23 3. Results and Discussion

24 3.1 Thin layer chromatography for the extract

7 1 The chromatographic profile of the H. banksii ethanol extract, visualised under visible light,

2 indicated the presence of 4 colourful bands (B1-B4) on the TLC plate. The light yellow (B1),

3 grey (B2) and green band (B3) on the TLC plate could correspond to some pigments as

4 carotene, pheophytin-like compounds and chlorophyll a, b, respectively. It has been reported

5 that ȕ- and Į-carotene was usually detected on the top of the TLC plate, followed by

6 xanthophylls and chlorophylls, while lutein/zeaxanthin was found at the bottom of the layer

7 (Jaime et al., 2005). Moreover, the dark grey band is likely pheophytin-like compounds that

8 appeared from the partial degradation process of chlorophyll a, b caused by the high

9 temperature or the long-time process of the extraction (Quach et al., 2004). The grey band

10 was observed to be small and not clear. It could be that a small amount of pheophytin-like

11 compounds appeared as the sample was evaluated immediately after extraction. The

12 fucoxanthin content (B4) with the colour of orange-yellow was separated to investigate

13 cytotoxic activity on pancreatic cancer cell lines.

14 3.2 Fucoxanthin content and isolation

15 The orange-yellow band (B4) from the H. banksii extract was detected at the wavelength of

16 450 nm and the retention time was at 3.95 min on HPLC system. Through TLC and HPLC

17 analysis compared with the standard commercial fucoxanthin, this band was confirmed to be

18 fucoxanthin (Figures 1, 2). Fucoxanthin and other pigments of H. banksii were visualised on

19 the TLC plate, while the quantitative data on fucoxanthin based on HPLC analysis was found

20 to be of 0.58 mg Fx.g-1 alga (dry weight). Fucoxanthin content was estimated by comparing

21 between the areas of the peaks of fucoxanthin and fucoxanthin added with the standard

22 (Figure 2). This finding was well fitted to fucoxanthin content (0.61 mg Fx.g-1 alga) that was

23 measured by reading the absorbance of the different wavelengths through a UV-Visible

24 spectrophotometer in our previous study (Dang et al., 2018).

8 1 Yield of fucoxanthin from brown algae varied greatly and depended on many factors such as

2 species, seasonality, geographic location, harvesting time, pre-treatment and extraction

3 methods. Fucoxanthin yield of the H banksii extract was in a range of fucoxanthin previously

4 reported. It was outlined that fucoxanthin content was 0.73 and 1.01 mg.g-1 in Sargassum

5 binderi and Sargassum duplicatum, respectively (Noviendri et al., 2011); the value of 0.39

6 mg.g-1 from Eisenia bicyclis (Kjellman) Setchell (Shang et al., 2011); Saccharina japonica

7 and Sargassum horneri with a range of 0.12–0.41 and 0.05–0.77 mg.g-1, respectively

8 (Sivagnanam et al., 2015). On the other hand, fucoxanthin content was found much higher in

9 micro-algae with 5.13 mg.g-1 in Chaetoceros calcitrans (Foo et al., 2017) and up to 18.23

10 mg.g-1 in Isochrysis aff. galbana (Kim et al., 2012).

11 Fucoxanthin was successfully isolated from the H.banksii extract via a chromatography

12 column using silica gel powder (Kieselgel 60GF254). The purity of fucoxanthin was obtained

13 to be 92.3%, and checked by HPLC analysis (Figure 2). The fucoxanthin isolated from this

14 specie was shown to be high purity at the first pass (>90% purity). In addition, the isolation

15 process of fucoxanthin was simple due to the long distances between the bands of the

16 pigments on the TLC plate with a proper mobile phase (Figure 1). The mixture of n-

17 hexane/acetone (7/3; v/v) was optimal for elution of fucoxanthin compared to the other ratios

18 or other solvents such as dichloromethane, ethyl acetate, ethanol or methanol (data not

19 shown). The results were supported by Jaswir et al. (2011) who reported that high purity of

20 fucoxanthin from alga Padina australis was 94.8% through the SiO2 chromatography column

21 (a mobile phase; n-hexane/acetone: 6/4 v/v) and up to 98.1% purity as the process was

22 repeated using a HPLC system. The purity of fucoxanthin >90% was also found in two

23 brown algae Sargassum binderi and Sargassum duplicatum using a silica column eluted with

24 a cold mixture of n-hexane/acetone: 6/4 (v/v) and acetone (Noviendri et al., 2011). However,

25 with the brown alga Undaria pinnatifida, fucoxanthin content was observed to be low purity

9 1 at the first fucoxanthin fraction (43.5%) and second fucoxanthin fraction (60.8%) via column

2 chromatography (Wang et al., 2014). The low purity of fucoxanthin obtained from algae may

3 be due to some reasons such as low ratio of fucoxanthin in the extract, the close distances

4 between fucoxanthin band and other compound bands (carotenoids, chlorophylls,

5 phenolics…) or/and inappropriate mobile phases for elution. It is clear that various algal

6 species, difference in components and their ratios within the extracts, resulted in different

7 efficacies in isolation of fucoxanthin. Moreover, almost all fucoxanthin content isolated from

8 brown algae was a type of all trans-fucoxanthin (Jaswir et al., 2013; Kim et al., 2011).

9 From the findings, fucoxanthin was directly obtained from the crude extract of H. banksii via

10 column chromatography with high purity. On the other hand, the crude algal extracts usually

11 were firstly partitioned to make fractions, and then fucoxanthin was eluted several times from

12 fucoxanthin enriched fractions (non-polar fractions) via column chromatography. For

13 example, fucoxanthin was obtained from the chloroform fraction of the Fucus evanescens

14 ethanol extract eluted using the mixture of benzene, n-hexane, ethyl acetate, chloroform and

15 ethanol (Imbs et al., 2013), from the dichloromethane fraction of the Scytosiphon lomentaria

16 extract (column chromatography with a mixed solvent (chloroform-acetone-methanol

17 100:10:1)/ethyl acetate 7:3 (v/v) as mobile phase; and then preparative TLC with

18 chloroform/ethyl acetate 7/3 (v/v) (Mori et al., 2004). Moreover, fucoxanthin could be

19 directly separated from the Himanthalia elongata extract using preparative thin layer

20 chromatography (TLC glass plate with mobile phase of chloroform/diethyl ether/n-

21 hexane/acetic acid (10:3:1:1, v/v/v/v)) (Rajauria et al., 2017). In addition, using centrifugal

22 partition chromatography (CPC) with a two-phase solvent system of n-hexane–ethyl acetate–

23 ethanol–water (5:5:7:3, v/v/v/v), fucoxanthin from Eisenia bicyclis (Kjellman) Setchell

24 (Laminariaceae) was separated in the system with the high purity (Kim et al., 2011). From the

25 findings, it is noted that brown alga H.banksii is considered as a good source for isolation of

10 1 fucoxanthin with simple procedure, high efficacy and significant quantity for further

2 applications.

3 3.3 Cytotoxic activity of fucoxanthin

4 The anti-cancer activity of fucoxanthin from the H.banksii extract was assessed against

5 pancreatic cancer cell lines (primary and secondary cell lines) as well as on non-tumourigenic

6 cells (HPDE) at serial concentrations (25-200 μg.mL-1). There was a significant difference in

7 cell growth inhibitory efficacies between low and high treated concentrations of fucoxanthin.

8 The results suggested that fucoxanthin inhibited the growth of pancreatic cancer cells in a

9 dose-dependent manner (Table 1).

10 In the case of MiaPaCa2 cells, there were significant differences in cell growth inhibition (%)

11 with the treatments of fucoxanthin at the different concentrations (P<0.05). High cell growth

12 LQKLELWLRQ ! E\IXFR[DQWKLQZDVREVHUYHGDWFRQFHQWUDWLRQV•—JP/-1. Cell growth

13 inhibition at 200 μg.mL-1 (92.81%) was significantly higher than that of gemcitabine 50 nM

14 (87.28%) (P<0.05). However, the cytotoxic activity towards the cancer cells was only 7.66%

15 at the treatment of 25 μg.mL-1. For the BxPC3 cell line, no significant difference of growth

16 inhibition was observed for the cells treated with fucoxanthin at 200, 150 μg.mL-1 and

17 gemcitabine 50 nM (P>0.05, Table 1). At the concentration of 100 μg.mL-1, 55.93% of

18 cancer cells were inhibited, while the cell growth inhibition was low (< 20%) at lower

19 FRQFHQWUDWLRQV ”—JP/-1). In regards to CFPAC1 cells, fucoxanthin at concentrations of

20 200, 150 and 100 μg.mL-1 inhibited 60.08, 47.7 and 30.91% of cell growth, respectively.

21 These values were lower compared to that of the standard (gemcitabine-50nM) with the

22 inhibition of 92.42%. With the non-tumorigenic cells (HPDE), it was found that fucoxanthin

23 presented medium cytotoxicity towards the cells with the inhibitions of 70.85 and 55.25% at

24 concentrations of 150 and 100 μg.mL-1, respectively. The proportion of 21.34% normal cells

11 1 was suppressed at the fucoxanthin of 25 μg.mL-1, while high cytotoxicity (89.67%) was

2 observed with the treatment of gemcitabine; 50 nM ~ 13.16x10-3 μg.ml-1.

3 Fucoxanthin showed potential efficacy against the pancreatic cancer cell lines as

4 characterised by the low IC50 values (Table 3). The IC50 value for MiaCaPa2 was 67.47

5 μg.mL-1, while these values for the BxPC3 and CFPAC1 cells were 97.68 and 166.31 μg.mL-

1 -1 6 , respectively. It was of 71.81μg.mL for non-tumourigenic HPDE cells. These IC50 values

7 showed significant inhibitory activity against pancreatic cancer cell lines with a moderate

8 toxicity towards the normal cells. Comparison to the cytotoxic activity of algae-derived

9 fucoxanthin, fucoxanthin from brown alga Padina australis showed low efficacy against

10 human lung cancer (H1299) with an IC50 value 2.45 mM (Jaswir et al., 2011). From Fucus

11 evanescens C Agardh, this pigment showed quite high anticancer activity (IC50 =114μM)

12 against human melanoma cell line (SK-MEL-28) (Imbs et al., 2013), while growth of the

13 melanoma cell line (B16F10) was inhibited by 87% upon 72 h exposure to 200 μM

14 fucoxanthin isolated from Ishige okamurae and fucoxanthin induced apoptosis was observed

15 via the presence of apoptotic bodies and nuclear condensation using the Hoechst 33342 stain

16 (Kim et al., 2013).

17 Fucoxanthin is hydrolysised to fucoxanthiol during intestinal absorption and partly converted

18 to amarouciaxanthin A in the liver (Maeda et al., 2006; Martin, 2015). Therefore, actions of

19 fucoxanthin against cancers were assessed via both fucoxanthin and its metabolites. The

20 mechanisms of these compounds are mediated through different signalling pathways,

21 including the caspases, Bcl-2 proteins, MAPK, PI3K/Akt, JAK/STAT, AP-1, GADD45, and

22 several other molecules that are involved in cell cycle arrest, apoptosis, anti-angiogenesis or

23 inhibition of metastasis (Martin, 2015). The roles of fucoxanthin as well as other carotenoids

24 against several cancers have been summarised in the previous studies (D’Orazio et al., 2012;

25 Kumar et al., 2013; Rengarajan et al., 2013; Tanaka et al., 2012).

12 1 There was little information about activities of fucoxanthin against pancreatic cancer

2 reported. It was shown that nitro-fucoxanthins (reaction products of fucoxanthin with

3 peroxinitrite) exhibited higher activity than fucoxanthin against pancreatic cancer cell line

4 (MiaCaPa-2) (Tsuboi et al., 2011). The algal extracts are the good source of fucoxanthin and

5 other compounds (phenolics and sulfated polysaccharides) against pancreatic cancer. Both

6 the Ulva sp. and H. banksii extracts had a selective cytotoxicity (high activity against cancer

7 cells and low toxicity towards normal ones) to a pancreatic cancer cell line (MiaCaPa2) with

8 no toxicity towards a normal murine cell line, while the nonpolar dichloromethane extracts

9 (the fucoxanthin-enriched fraction) of Phyllospora comosa, Solieria robusta and Ulva sp.

10 showed high cytotoxic activity (76-100%) on MiaCaPa2 cells at 100 μg.mL-1 (McCauley et

11 al., 2015). The cytotoxicity of the Fucus vesiculosus acetone extract (Fv1) was evaluated on

12 pancreatic cancer cell lines (panc1; panc98; pancTU1 and Colo357). It showed that Fv1

13 inhibited strongly the growth of different tumour cell lines with the EC50 values of 17.35

14 ȝJP/-1 for PancTU1; 17.5 ȝJP/-1 for Panc89; 19.23 ȝJP/-1 for Panc1 and 28.9 ȝJP/-1

15 for Colo357. Fv1 showed low cytotoxic activity against non-malignant resting T cells

16 (Geisen et al., 2015). As discussed above, only crude algal extracts against pancreatic cancer

17 cell lines were reported. These extracts indicated high efficacy against pancreatic cancer cell

18 lines. However, it was hard to determine which compounds fucoxanthin, phenolics,

19 polysaccharides or others with main roles against pancreatic cancer. In our work with the

20 H.banksii extract, phenolics showed higher inhibition for pancreatic cancer cell lines than

21 fucoxanthin, but higher toxicity towards normal cells compared to that of fucoxanthin, while

22 polysaccharides with significant cytotoxic effect against pancreatic cancer were safety for

23 normal cells (data not shown).

24 The selective cytotoxicity of fucoxanthin was demonstrated by the previous studies. Wang et

25 al. (Wang et al., 2014) reported that in three non-tumourigenic cell lines, the HUVEC cell

13 1 line was the most sensitive to fucoxanthin (IC50= 4.42 μM) after 72 h treatment, while the

2 growth of HDFB (IC50= 21.05 μM)andHEK293(IC50= 13.48 μM) cells were less affected

3 by fucoxanthin at concentrations lower than IC50 value. In addition, it has been shown that

4 HUC-Fm (human male umbilical cord fibroblast) and MRC-5 (human normal embryonic

5 lung fibroblast) cells were unaffected by the treatment with fucoxanthin at 5 μM (Kotake-

6 Nara et al., 2005). Importantly, it was shown that fucoxanthin showed toxic effects on non-

7 malignant cells in cell culture, but had no toxic effects in vivo testes (Geisen et al., 2015). The

8 pigment was safe for mice fed with a single dose of 1000-2000 mg/kg, or repeated doses of

9 500-1000 mg/kg for 30 days. No fatalities or abnormalities were found in fucoxanthin treated

10 groups (Beppu et al., 2009). Therefore, potential of algal-derived fucoxanthin against cancers

11 is needed in order to assess more through in vivo tests and further clinical use.

12 From the findings and the reports of previous studies in literature, fucoxanthin from

13 H.banksii with high cytotoxic activity against pancreatic cancer indicated potential for further

14 pharmaceutical applications. In further studies, the mechanism of actions by fucoxanthin

15 against the pancreatic cancer cell lines will be investigated. In addition, combination of

16 fucoxanthin and other compounds (gemcitabine, algal phenolics, sulfated polysaccharides)

17 will be evaluated for selective cytotoxicity against pancreatic cancer in vitro and in vivo tests.

18 4. Conclusions

19 The isolation of fucoxanthin from the brown alga H.banksii has been successfully

20 demonstrated at the laboratory-scale. The purified pigment was confirmed to be fucoxanthin

21 by comparing with a standard fucoxanthin through TLC and HPLC analysis. The fucoxanthin

22 content in the alga was of 0.58 mg Fx.g-1 alga and the purity of this compound achieved

23 92.3% using a chromatography column. Fucoxanthin showed high anti-cancer activity on

24 pancreatic cancer cell lines compared to the gemcitabine, and moderate cytotoxicity towards

14 1 non-tumourigenic cells. The H. banksii extract-derived fucoxanthin has potential to be used

2 as a treatment of pancreatic cancer and further applications in pharmaceutical field.

3

4

5

6

7 Acknowledgements

8 The authors also kindly thank to University of Newcastle; the Vietnamese Government

9 through the Ministry of Education and Training; the Ministry of Agriculture and Rural

10 Development for awarding a VIED-TUIT scholarship to Thanh Trung DANG.

11

12

13

14

15

16

17 Conflict of interest

18 The authors declare no conflict of interest.

19

20

21

22

23

24

25

15 1 References

2 Balboa, E. M., Conde, E., Moure, A., Falqué, E., & Domínguez, H., 2013. In vitro

3 antioxidant properties of crude extracts and compounds from brown algae. Food Chem.

4 138 (2), 1764-1785.

5 Behrens, D., Walther, W., Fichtner, I., 2017. Pancreatic cancer models for translational

6 research. Pharmacol. Therapeut.173, 146-158.

7 Beppu, F., Niwano, Y., Tsukui, T., Hosokawa, M., Miyashita, K., 2009. Single and repeated

8 oral dose toxicity study of fucoxanthin (FX), a marine carotenoid, in mice. J. Toxicol

9 Sci. 34(5), 501-510.

10 D’Orazio, N., Gemello, E., Gammone, M. A., de Girolamo, M., Ficoneri, C., Riccioni, G.,

11 2012. Fucoxantin: A treasure from the sea. Mar. Drugs 10 (3), 604-616.

12 Dang, T. T., Bowyer, M. C., Van Altena, I. A., Scarlett, C. J., 2018. Comparison of chemical

13 profile and antioxidant properties of the brown algae. Inter. J. Food Sci. Technol. 53 (1),

14 174-181.

15 Foo, S. C., Yusoff, F. M., Ismail, M., Basri, M., Yau, S. K., Khong, N. M., Ebrahimi, M.,

16 2017. Antioxidant capacities of fucoxanthin-producing algae as influenced by their

17 carotenoid and phenolic contents. J. Biotechnol. 241, 175-183.

18 Fung, A., Hamid, N., Lu, J., 2013. Fucoxanthin content and antioxidant properties of Undaria

19 pinnatifida. Food Chem. 136(2), 1055-1062.

20 Geisen, U., Zenthoefer, M., Peipp, M., Kerber, J., Plenge, J., Managò, A., Adam, D., 2015.

21 Molecular mechanisms by which a Fucus vesiculosus extract mediates cell cycle

22 inhibition and cell death in pancreatic cancer cells. Mar. Drugs 13 (7), 4470-4491.

23 Gupta, S., Abu-Ghannam, N., 2011. Bioactive potential and possible health effects of edible

24 brown seaweeds. Trend Food Sci. Technol. 22(6), 315-326.

16 1 Imbs, T.I., Ermakova, S. P., Fedoreyev, S. A., Anastyuk, S. D., Zvyagintseva, T. N., 2013.

2 Isolation of fucoxanthin and highly unsaturated monogalactosyldiacylglycerol from

3 brown alga Fucus evanescens C Agardh and in vitro investigation of their antitumor

4 activity. Mar. Biotechnol. 15 (5), 606-612.

5 Jaime, L., Mendiola, J.A., Herrero, M., Soler-Rivas, C., Santoyo, S., Señorans, F.J., Ibáñez,

6 E., 2005. Separation and characterization of antioxidants from Spirulina platensis

7 microalga combining pressurized liquid extraction, TLC, and HPLC-DAD. J. Sep. Sci.

8 28 (16), 2111-2119.

9 Jaswir, I., Noviendri, D., Salleh, H.M., Taher, M., Miyashita, K., 2011. Isolation of

10 fucoxanthin and fatty acids analysis of Padina australis and cytotoxic effect of

11 fucoxanthin on human lung cancer (H1299) cell lines. African J. Biotechnol. 10 (81),

12 18855-18862.

13 Jaswir, I., Noviendri, D., Salleh, H.M., Taher, M., Miyashita, K., Ramli, N., 2013. Analysis

14 of fucoxanthin content and purification of all-trans-fucoxanthin from Turbinaria

15 turbinata and Sargassum plagyophyllum by SiO2 open column chromatography and

16 reversed phase-HPLC. J. Liq. Chromatogr. R.T. 36(10), 1340-1354.

17 Kim, K.N., Ahn, G., Heo, S.J., Kang, S.M., Kang, M.C., Yang, H.M., Jeon, B.T., 2013.

18 Inhibition of tumor growth in vitro and in vivo by fucoxanthin against melanoma

19 B16F10 cells. Environ. Toxicol. Pharm. 35(1), 39-46.

20 Kim, S. M., Kang, S.W., Kwon, O.N., Chung, D., Pan, C.H., 2012. Fucoxanthin as a major

21 carotenoid in Isochrysis aff. galbana: Characterization of extraction for commercial

22 application. J. Korean Soc. Appl. Bi. 55(4), 477-483.

23 Kim, S. M., Shang, Y. F., & Um, B. H., 2011. A preparative method for isolation of

24 fucoxanthin from Eisenia bicyclis by centrifugal partition chromatography. Phytochem

25 Analysis 22(4), 322-329.

17 1 Kotake-Nara, E., Sugawara, T., Nagao, A., 2005. Antiproliferative effect of neoxanthin and

2 fucoxanthin on cultured cells. Fish Sci. 71(2), 459-461.

3 Kumar, S. R., Hosokawa, M., Miyashita, K., 2013. Fucoxanthin: A Marine Carotenoid

4 Exerting Anti-Cancer Effects by Affecting Multiple Mechanisms. Mar. Drugs 11(12),

5 5130-5147.

6 Maeda, H., Hosokawa, M., Sashima, T., Takahashi, N., Kawada, T., Miyashita, K., 2006.

7 Fucoxanthin and its metabolite, fucoxanthinol, suppress adipocyte differentiation in 3T3-

8 L1 cells. Inter. J. Mol. Med. 18(1), 147-152.

9 Martin, L. J., 2015. Fucoxanthin and its metabolite fucoxanthinol in cancer prevention and

10 treatment. Mar. Drugs 13(8), 4784-4798.

11 McCauley, J. I., Meyer, B. J., Winberg, P. C., Ranson, M., Skropeta, D., 2015. Selecting

12 Australian marine macroalgae based on the fatty acid composition and anti-inflammatory

13 activity. J. Appl. Phycol. 27(5), 2111-2121.

14 Mohamed, S., Hashim, S. N., Rahman, H. A., 2012. Seaweeds: a sustainable functional food

15 for complementary and alternative therapy. Trend Food Sci. Technol. 23(2), 83-96.

16 Mori, K., Ooi, T., Hiraoka, M., Oka, N., Hamada, H., Tamura, M., Kusumi, T., 2004.

17 Fucoxanthin and its metabolites in edible brown algae cultivated in deep seawater. Mar.

18 Drugs 2(2), 63-72.

19 Nakazawa, Y., Sashima, T., Hosokawa, M., Miyashita, K. (2009). Comparative evaluation of

20 growth inhibitory effect of stereoisomers of fucoxanthin in human cancer cell lines. J.

21 Funct Food. 1(1), 88-97.

22 Noviendri, D., Jaswir, I., Salleh, H.M., Taher, M., Miyashita, K., Ramli, N., 2011.

23 Fucoxanthin extraction and fatty acid analysis of Sargassum binderi and S. duplicatum.

24 J. Med. Plant Res. 11, 2405-2412.

18 1 Peng, J., Yuan, J.P., Wu, C.F., Wang, J.H., 2011. Fucoxanthin, a marine carotenoid present in

2 brown seaweeds and diatoms: metabolism and bioactivities relevant to human health.

3 Mar. Drugs 9(10), 1806-1828.

4 Petrelli, F., Inno, A., Ghidini, A., Rimassa, L., Tomasello, G., Labianca, R., Barni, S., 2017.

5 Second line with oxaliplatin-or irinotecan-based chemotherapy for gemcitabine-

6 pretreated pancreatic cancer: A systematic review. European J. Cancer 81, 174-182.

7 Quach, H.T., Steeper, R.L., Griffin, G.W., 2004. An improved method for the extraction and

8 thin-layer chromatography of chlorophyll a and b from spinach. J. Chem. Educ 81(3),

9 385.

10 Rajauria, G., Foley, B., Abu-Ghannam, N., 2017. Characterization of dietary fucoxanthin

11 from Himanthalia elongata brown seaweed. Food Res. Inter. 99 (3), 995-1001

12 Raviv, N. V., Sakhuja, S., Schlachter, M., Akinyemiju, T., 2017. Metabolic syndrome and in-

13 hospital outcomes among pancreatic cancer patients. Diabete Metab. Syndr: Clin. Res.

14 Rev. 11 (2), S643-S650.

15 Rengarajan, T., Rajendran, P., Nandakumar, N., Balasubramanian, M. P., Nishigaki, I., 2013.

16 Cancer preventive efficacy of marine carotenoid fucoxanthin: cell cycle arrest and

17 apoptosis. Nutrients 5(12), 4978-4989.

18 Scarlett, C. J., Salisbury, E. L., Biankin, A. V., Kench, J., 2011. Precursor lesions in

19 pancreatic cancer: morphological and molecular pathology. Pathol. 43(3), 183-200.

20 Schumacher, M., Kelkel, M., Dicato, M., Diederich, M., 2011. Gold from the sea: marine

21 compounds as inhibitors of the hallmarks of cancer. Biotechnol Adv. 29(5), 531-547.

22 Shang, Y.F., Kim, S.M., Lee, W.J., Um, B.H., 2011. Pressurized liquid method for

23 fucoxanthin extraction from Eisenia bicyclis (Kjellman) Setchell. J. Bioscie. Bioeng.

24 111(2), 237-241.

19 1 Sithranga Boopathy, N., Kathiresan, K., 2010. Anticancer drugs from marine flora: an

2 overview. J Oncol. 2010. doi.org/10.1155/2010/214186

3 Sivagnanam, S.P., Yin, S., Choi, J.H., Park, Y. B., Woo, H. C., Chun, B. S., 2015. Biological

4 properties of fucoxanthin in oil recovered from two brown seaweeds using supercritical

5 CO2 extraction. Mar. Drugs 13(6), 3422-3442.

6 Tanaka, T., Shnimizu, M., & Moriwaki, H., 2012. Cancer chemoprevention by carotenoids.

7 Molecules 17(3), 3202-3242.

8 Terasaki, M., Hirose, A., Narayan, B., Baba, Y., Kawagoe, C., Yasui, H., Miyashita, K.

9 (2009). Evaluation of recoverable functional lipid components of several brown

10 seaweeds (Phaeophyta) from Japan with special reference to fucoxanthin and fucosterol

11 contents. J. Phycol. 45(4), 974-980.

12 Tsuboi, M., Etoh, H., Kato, K., Nakatugawa, H., Kato, H., Maejima, Y., Miyashita, K., 2011.

13 Nitrocapsanthin and nitrofucoxanthin, respective products of capsanthin and fucoxanthin

14 reaction with peroxynitrite. J. Agric. Food Chem. 59(19), 10572-10578.

15 Wang, S. K., Li, Y., White, W. L., & Lu, J., 2014. Extracts from New Zealand Undaria

16 pinnatifida containing fucoxanthin as potential functional biomaterials against cancer in

17 vitro. J. Funct Biomater. 5(2), 29-42.

18 Wijesinghe, W., & Jeon, Y.J., 2012. Exploiting biological activities of brown seaweed

19 Ecklonia cava for potential industrial applications: a review. Inter. J. Food Sci. Nutr.

20 63(2), 225-235.

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20 1 Figure Captions

2 Fig. 1: TLC for the extract and purified fucoxanthin (PF) compared with the standard 3 commercial fucoxanthin (Std). B1-B4 represent for the bands of the pigments

4 Fig. 2: HPLC chromatogram of the purified fucoxanthin (top) and purified fucoxanthin 5 added to the standard fucoxanthin (bottom), and structure of all-trans Fucoxanthin 6

21 Table

Table 1. The cytotoxic activity (percentage of cancer cell growth inhibition; %) of the purified fucoxanthin at different concentrations in pancreatic cancer cell lines Concentration of the fucoxanthin (μg.mL-1) and gemcitabine (50 nM) Cell lines 200 150 100 50 25 Gem. HDPE 82.72±3.39a 70.85±2.92b 55.25±4.19c 39.29±3.38d 21.34±4.89e 89.67±0.98f MiaCaPa2 92.81±1.72a 84.5±2.52b 75.9±2.95c 31.73±3.78d 7.66±4.65e 87.28±1.59b BxPC3 71.71±5.81a 70.3±4.53a 55.93±4.97b 14.62±2.09c 6.74±2.92d 75.03±1.82a CFPAC1 60.08±4.17a 47.70±3.61b 30.91±1.46c 14.48±3.32d 6.52±1.88e 92.42±2.34f All values are means ± standard deviation (n=6) and those in the same row not sharing the same superscript letter are significantly different from the others (P<0.05). Table 2. Value IC50 - The concentration of fucoxanthin purified from brown alga H.banksii that inhibits cell growth by 50% for normal and pancreatic cell lines. -1 Cell lines IC50 Value (μg.mL ) HDPE 71.81 MiaCaPa2 67.47 BxPC3 97.68 CFPAC1 166.31 Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Paper VII:

“Antioxidant and cytotoxic activity (in vitro) of phlorotannin-enriched fractions from the brown alga Hormosira banksii (Turner) Decaisne”. Submitted to Journal of Marine Biotechnology.

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Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation 1 Antioxidant and cytotoxic activities (in vitro) of phlorotannin-enriched fractions from 2 brown alga Hormosira banksii (Turner) Decaisne

3 Thanh T. Dangabd*, Jennette A. Sakoff c, Michael C. Bowyera, Ian A. Van Altenaa & Christopher J. Scarlettab 4

5 a School of Environmental and Life Sciences, University of Newcastle, Ourimbah, NSW, 2258 Australia 6 b Pancreatic Cancer Research Group, University of Newcastle, 10 Chittaway Rd, Ourimbah, 2258 Australia 7 c Department of Medical Oncology, Calvari Mater, Newcastle Hospital, Warratah, NSW, Australia 8 d Department of Seafood Processing Technology, Nha Trang University, Khanh Hoa, Vietnam

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17 Running Title: Phlorotannins - enriched fractions and their anticancer activity.

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20 *Corresponding author: 21 Thanh Trung Dang 22 School of Environmental and Life Sciences, Faculty of Science, University of Newcastle. 23 10 Chittaway Rd, Ourimbah, NSW 2258, Australia. 24 Ph: +61 458926707 Fax: +61 2 4348 4145 25 Email: [email protected]

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1 1 Abstract

2 Brown algae are a rich source of bioactive compounds, especially phenolics that have potential for the treatment

3 of several cancers. This study aimed to evaluate the antioxidant and cytotoxic activities of the phlorotannin-

4 enriched fractions from the brown algae Hormosira banksii on several cancer cell lines (focussing on pancreatic

5 cancer cells). The results revealed that the phlorotannins (in the ethyl acetate (EA) and butanol (BuOH)

6 fractions) were mainly responsible for antioxidant activities of the extract. These fractions strongly inhibited

7 (the inhibition > 80% in almost all cell lines) cell growth on several cancer cell lines. For pancreatic cancer, the

-1 -1 8 EA fraction (IC50 = 29.08 μg.mL ) suppressed MiaCaPa2 by 79.42% at the low concentration of 50 μg.mL ,

-1 -1 9 while the BuOH fraction (IC50 = 58.06 μg.mL ) showed high growth inhibition of 79.14% at 100 μg.mL .

10 However, the medium-polar phenolics (the EA fraction) were found to be highly toxic towards the normal cells

-1 11 with an IC50 value of 1.72 μg.mL . Interestingly, the polar phenolics (the BuOH fraction) showed selective

-1 12 anticancer activity with an IC50 value of 106.45 μg.mL for the normal cells. Phlorotannin-enriched fractions

13 showed impressive growth inhibition efficacy against pancreatic cancer cell lines and further investigations

14 should be conducted for delineation of their effects on apoptotic and cell cycle mechanisms, as well as the

15 potential applications in the pharmaceuticalfields.

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17 Keyword: Phlorotannins; Hormosira banksii; Antioxidant Activity; Pancreatic Cancer.

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2 1 Introduction

2 Marine algae have been increasingly investigated in the search for bioactive compounds to develop new drugs

3 and healthy foods (El Gamal 2010). Edible marine macroalgae (seaweeds) are classified into three divisions;

4 Chorophyta (green algae), Phaeophyta (brown algae), and Rhodophyta (red algae) according to their

5 composition of pigments and their chemical composition (Gupta and Abu-Ghannam 2011). Brown algae

6 (Phaeophyta) accumulate a variety of phloroglucinol-based polyphenols (called phlorotannins). Phlorotannins

7 are formed by the polymerization of phloroglucinol (1,3,5-trihydroxybenzene) monomer units and are highly

8 hydrophilic components with a wide range of molecular sizes ranging between 126 kDa and 650 kDa (Eom et

9 al. 2012). Based on the means of linkage, phlorotannins can be classified into four subclasses; fuhalols and

10 phlorethols (phlorotannins with an ether linkage), fucols (with a phenyl linkage), fucophloroethols (with an

11 ether and phenyl linkage), and eckols, carmalols (with a dibenzodioxin linkage) (Singh and Bharate 2006). It has

12 been shown that phlorotannins are found abundantly in brown algae, and that brown algae had a higher

13 antioxidant capacity than those of red or green algae (Balboa et al. 2013). In addition, it has been demonstrated

14 that the compounds in algal extracts have been linked to treatment of some chronic diseases (anti-diabetes, anti-

15 inflammatory, radiation protection and anti-allergic activities), as well as several types of cancers (Schumacher

16 et al. 2011; Sithranga Boopathy and Kathiresan 2010).

17 Pancreatic cancer is one of the leading causes of cancer death in the Western countries (Behrens et al. 2017;

18 Scarlett et al. 2011), and despite significantly improved understanding of its biology and pathogenesis, current

19 treatments are found to be insufficient with mortality caused by pancreatic cancer closely paralleling its

20 incidence. Therapeutic options applicable to pancreatic cancer are still mainly limited to surgical resection,

21 while chemotherapy and radiotherapy are applied to almost all pancreatic cancer patients with limited efficacy

22 (Raviv et al. 2017). Gemcitabine (2,2-difluoro 2-deoxycytidine), the first-line therapy for pancreatic cancer is

23 inadequate due to its high toxicity and drug resistance, while the combination of gemcitabine with albumin-

24 bound paclitaxel particles (nab-paclitaxel) or FOLFIRINOX (the combination of Folinic acid, 5-Fluorouracil(5-

25 FU), Irinotecan, and Oxaliplatin) have also marginally increased overall survival for a small subset ofpancreatic

26 cancer patients when compared to gemcitabine alone (Grasso et al. 2017). With the limited therapeutic options

27 for pancreatic cancer, the biologically active components within the algal extracts may be potentially useful as

28 lead compounds for further drug development.

29 There is a lack of information on the components of H. banksii extracts and the treatment of algal-derived

30 compounds on cancer cells in vitro. Therefore, this study aimed to isolate phlorotannins from the alga H. banksii

3 1 and the phlorotannin-enriched fractions were evaluated for their cytotoxic activity on several cancer cell lines

2 (more detailed in pancreatic cancer cells).

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4 Materials and methods

5 Materials

6 The alga H. banksii was collected in March, 2016 from the rocky shore at Bateau Bay, NSW, Australia

7 (Latitude of 33°22'55.2"S; longitude of 151°29'6"E). After collection, the sample was washed with seawater and

8 then washed thoroughly with fresh water, freeze dried for 48 h using a freeze dryer. The dried alga was

9 shredded, sieved ” 600 μm particle size) and stored at – 20 °C for further analysis.

10 Preparation of the phlorotannin-enrichedfractions

11 The freeze dried alga was extracted with ethanol (70%) using an ultrasonic bath (Soniclean, 220 V, 50 Hz and

12 250W, Soniclean Pty Ltd, Australia) set at temperature of 30 °C, time 60 min and power of 150 watts as

13 described by Dang et al. (2017a). The extract was filtered and the filtrate was concentrated at temperature of 35

14 °C using a rotary evaporator (Buchi Rotavapor B-480, Buchi, Australia), then freeze dried to obtain the dried

15 extract. The extract was re-dissolved in water and partitioned with n-hexane (Hx), dichloromethane (DCM),

16 ethyl acetate (EA), and butanol (BuOH). The crude extract and five fractions were obtained (Hx, DCM, EA,

17 BuOH and an aqueous fraction (AQ)) and stored at – 20 °C beforeanalysis.

18 Thin layer chromatography (TLC) analysis for fractions

19 Phenolic compounds in EA, BuOH and AQ fraction could be observed through vanillin stain with a mixture of

20 CHCl3/methanol/water/acetic acid (50:25:4:3 v/v/v/v) as the mobile phase (Shibata et al. 2002)

21 Total phenolic content

22 Total phenolic content (TPC) was conducted as described by Skerget et al. (2005) with slight modifications.The

23 absorbance was measured at 765 nm using a UV spectrophotometer (Varian Australia Pty. Ltd., Victoria,

24 Australia). Gallic acid was used as a standard and the results were expressed as mg of gallic acid equivalentsper

25 gram of the sample (mg GAE.g-1).

26 Determination of antioxidant capacity

27 ABTS total antioxidant capacity (ABTS) was measured as described by Thaipong et al. (2006). Methanol and

28 trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid) were used as the control and standard,

29 respectively. The absorbance was measured at 734 nm and the results were expressed as mg of trolox

30 equivalents per gram of the sample (mg TE.g-1).

4 1 DPPH Free radical scavenging capacity (DPPH): The extract was analysed as previously described by Brand-

2 Williams et al. (1995). Trolox was used as a standard and the colour of the sample was read at 515nm. The

3 results were expressed as mg TE.g-1 the sample (mg TE.g-1).

4 Ferric reducing antioxidant power (FRAP): The extract was measured as described by Benzie and Strain

5 (1999). Trolox was used as a standard and the sample was measured at 593nm. The results were expressed as

6 mg TE.g-1 the sample (mg TE.g-1).

7 Cytotoxic activities of the fractions on cancer celllines

8 The cytotoxicity of the algal fractions and crude extract was evaluated using the MTT (3-(4,5-dimethylthiazol-2-

9 yl)-2,5-diphenyltetrazolium bromide) assay on several cancer cell lines in vitro including HT29 (colon); U87,

10 SJ-G2, SMA (glioblastoma); MCF-7 (breast); A2780 (ovarian); H460 (lung); A431 (skin); Du145 (prostate);

11 BE2-C (neuroblastoma); MiaPaCa2 (pancreas) and the normal breast cell line (MCF10A) as previously

12 described by Chuen et al. (2016). All cancer cell lines were cultured in Dulbecco’s Modified Eagle Medium

13 (DMEM) supplemented with 10% foetal bovine serum, 50IU.mL-1 penicillin, 50 mg.mL-1 streptomycin and 2

14 mM L-glutamine. The MCF10A cells were cultured in DMEM:F12 (1:1) cell culture media, 5% heatinactivated

15 horse serum, supplemented with penicillin (50 IU.mL-1), streptomycin (50 mg.mL-1), 20 mM Hepes, L-

16 glutamine (2 mM), epidermal growth factor (20 ng.mL-1), hydrocortisone (500 ng.mL-1), cholera toxin (100

17 ng.mL-1), and insulin (10 ug.mL-1). Cells were cultured in a 96 well plate with the density of 2500–4000 cells

18 per well. The cells were treated by the fractions and crude extract at the concentration of 100 μg.mL-1 for 72 h

19 and the absorbance was read at 540 nm using a multi-well spectrophotometer (BIORAD Benchmark PlusTM).

20 The cytotoxicity of the fractions was determined as a percentage of dead cells compared to the control (DMSO)

21 (% inhibition = ((Abs of the control – Abs of the sample)/Abs of the control x 100% and each sample was

22 conducted at least in triplicate (n=3-6).

23 Determination of cytotoxic activity on pancreatic cancer celllines

24 Cell culture

25 Human pancreatic cancer cells (two primary pancreatic cancer cell lines: MiaPaCa2, BxPC3 and one secondary

26 pancreatic cancer cell line: CFPAC1) and human pancreatic ductal epithelial cells (HPDE) were cultured at 37

27 °C, 5% CO2. Keratinocyte Serum-free Media (K-SFM) with human Recombinant Epidermal Growth Factor

28 (EGF) and Bovine Pituitary Extract (BPE) (10%) were used for HPDE. Dulbecco’s Modified Eagle’s Medium

29 (DMEM) supplemented with 10% foetal bovine serum (FBS), 2.5% horse serum and L-glutamine (100μg.mL-1)

5 1 were used to culture Mia-PaCa-2 cells, while 10% FBS and L-glutamine (100 μg.mL-1)inRPMImediawere

2 applied for BxPC3 and IMDM media for CFPAC1.

3 Cytotoxic activity of the phlorotannin-enrichedfractions

4 Cytotoxic activity of the phlorotannin-enriched fractions was determined using the Dojindo Cell Counting Kit-8

5 (CCK-8: Dojindo Molecular Technologies, Inc., Rockville, MD, USA) as described by Bhuyan et al. (2017).

6 The cells (200 μL) were seeded into a 96-well plate at 3x103 cells per well for MiaCaPa2 and 7x103 cells for

7 HPDE, BxPC3 and CFPAC1 and incubated to adhere for 24 h. The cells were then treated with various

8 concentrations (25-200 μg.mL-1) of the fractions, gemcitabine (50 n M; ~13,16 ng.ml-1); and 0.5% DMSO.

9 After 72 h, 10 μL of CCK-8 solution was added before incubating at 37 °C with 5% CO2 for 90 min. The

10 absorbance was measured at 450 nm on a multi-well spectrophotometer (BIORAD Benchmark PlusTM), and the

11 cytotoxic activity of the fractions was determined as a percentage of dead cells compared to the control

12 (DMSO). All experiments were performed in replicates of6.

13 Statistical Analysis

14 A one-way ANOVA and LSD post-hoc test were employed (SPSS Statistical Software, Version 16) to analyse

15 the differences between the independent samples. Differences between the mean levels of analyses were taken

16 to be statistically significant at P<0.05. All experiments were conducted at least in triplicate and the results were

17 performed as means ± standard deviations. The IC50 (the concentration required inhibits cell growth by 50%)

18 values were calculated by curve fitting the absorbance (viability) vs. log [concentration of treatment] using

19 GraphPad Prism software (version 7.03).

20 Results and Discussion

21 Thin layer chromatography (TLC) analysis for fractions

22 The spots of the EA, BuOH and AQ fraction were visualized under visible light by spraying with the vanillin-

23 H2SO4 reagent. The formation of red bands following spraying with vanillin in an ethanolic sulfuric acid

24 solution, and heating on the TLC plate confirmed that compounds from the EA, BuOH and AQ fraction were

25 phenolic (called phlorotannins in marine species) (Kubanek et al. 2004; Martínez and Castañeda 2013) (Figure

26 1). From the TLC analysis, it is well fitted with the findings in Figure 2 that there was a small amount of the

27 polar phenolic compounds in the AQ fraction. Phenolic compounds (medium polarity) had a large proportion in

28 the EA fraction, while a high ratio of polar phenolics was found in the BuOH fraction. Currently, the isolation

29 and structural identification of the active compounds of the EA and BuOH fraction are being employed using a

6 1 chromatography column, liquid chromatography–mass spectrometry (LC/MS) and nuclear magnetic resonance

2 (NMR).

3 Total phenolic content (TPC)

4 As shown in Figure 2, the TPC values of the crude extract (CE) and phlorotannin-enriched fractions (the EAand

5 BuOH fraction) were evaluated. The EA fraction had the highest TPC (473.74 mg GAE.g-1), three-fold higher

6 than the CE (158.82 mg GAE.g-1). The high value of TPC (277.69 mg GAE.g-1) was also observed in the BuOH

7 fraction, nearly two-fold higher than the CE, while it was only 37.95 mg GAE.g-1 for the AQ fraction. From the

8 findings, the significant differences in TPC among the fractions implied that isolation of the phenolic

9 compounds from algae by solvents with different polarities was efficient; and ethyl acetate and butanol were the

10 solvent of choice for separation of phenolic compounds in the alga H. banksii. In agreement with the literature,

11 TPC was also demonstrated to be the highest in the EA fraction of various algae such as Turbinaria conoides

12 and Turbinaria ornata (Chakraborty et al. 2013), Rhodomela confervoides (Rhodomelaceae) (Wang et al.

13 2009a), Fucus vesiculosus (Wang et al. 2012) and Polysiphonia urceolata (Duan et al. 2006). Conversely, the

14 most phenolic compounds were found in the AQ fractions prepared from Sargassum marginatum, Padina

15 tetrastomatica and Turbinaria conoides (Chandini et al. 2008), while petroleum ether was best for extracting

16 phenolic compounds from Gracilaria edulis and Acanthophora spicifera (Ganesan et al. 2008). The differences

17 in TPC values in the fractions were due to the difference of algal species and environmental conditions in where

18 they grow. The varying algal species and environmental conditions also resulted in the differences in both the

19 constituents and the ratio of components in the algae (Cho et al.2010).

20 Almost all phenolics, with high antioxidant activity found in the EA fractions from brown algae, were

21 continuously isolated using column chromatography, identified by comparing their LC/MS (liquid

22 chromatography/mass spectrophotometry) or NMR (nuclear magnetic resonance) data to the literature data, and

23 assessed for anticancer activity. For example, phloroglucinol, eckol, dioxinodehydroecko, and dieckol were

24 isolated from the Eisenia bicyclis extract (Kang et al. 2013); four phlorotannins as eckol, 2-phloroeckol,

25 phlorofucofuroeckol B, and ƍ-bieckol from Ecklonia stolonifera (Lee et al. 2012);

26 diphlorethohydroxycarmalol from Ishige okamurae (Kang et al. 2012) and dieckol from Ecklonia stolonifera

27 (Yoon et al. 2013).

28 Antioxidant capacity of the crude extract and fractions

29 Three assays were performed to evaluate antioxidant activity of the crude extract and the phlorotannin-enriched

30 fractions at the concentration of 0.12 mg.mL-1 (the serial concentrations of the extract and fractions: 0.06; 0.12;

7 1 0.25; 0.5 and 1 mg.mL-1 were tested for antioxidant activities (data not shown). The concentration of 0.12

2 mg.mL-1 was published because it is close to that was tested for the cell growth inhibition). It can be seen from

3 Figure 3 that the EA fraction performed the best in all antioxidant assays compared with the crude extract and

4 the BuOH fraction. Of note, the total antioxidant capacity (ABTS) of EA fraction (143.28 mg TE.g-1)was

5 significantly higher than that of the positive controls: BHT (butylated hydroxytoluene; 132.42 mg TE.g-1) andĮ-

6 tocopherol (97.25 mg TE.g-1) (P<0.05), and comparable to ascorbic acid (165.16 mg TE.g-1). The medium-polar

7 phenolic compounds were mainly responsible for the antioxidant activity. The BuOH fraction also had high

8 ABTS activity (78.39 mg TE.g-1), as such butanol and ethyl acetate were the optimal solvents for extraction of

9 the phenolic compounds with high antioxidant activity from brown alga H. banksii. Similar to the trend of the

10 ABTS activity, the highest DPPH radical scavenging capacity was found in the EA fraction (121.88 mg TE.g-1).

11 The EA fraction had a DPPH value significantly higher than BHT and Į-tocopherol, while the DPPH value of

12 the BuOH fraction (56.96 mg TE.g-1) was significantly higher than that of BHT (P<0.05). These data support

13 the findings in the literature whereby the greatest DPPH values were observed in the EA fraction of several

14 brown algae: Polysiphonia urceolata, Sargassum marginatum, Rhodomela confervoides and Turbinaria ornata

15 (Chakraborty et al. 2013; Chandini et al. 2008; Duan et al. 2006; Wang et al. 2009b). In comparison to the

16 positive controls, Cho et al. (2011) reported that the highest DPPH in the chloroform fraction from green alga

17 Enteromorpha prolifera was much lower than that of BHA at the concentrations of 0.06 and 0.13 mg.mL-1. All

18 the organic fractions from red alga Polysiphonia urceolata were lower compared to gallic acid and ascorbic acid

19 at all concentrations investigated (Duan et al. 2006). For the red alga Rhodomela confervoides, at low

20 concentrations (0.4, 2, and 10 μg.mL-1) the EA fraction had DPPH values much lower than ascorbic acid and

21 gallic acid (Wang et al. 2009a). The DPPH value of the extracts and fractions from three brown algae

22 Sargassum marginatum, Padina tetrastomatica and Turbinaria conoides were also reportedly lower than that of

23 Į-tocopherol at 1 mg.mL-1 (Chandini et al. 2008). However, in FRAP assay, the FRAP values of the crude

24 extract (8.17 mg TE.g-1) and fractions (5.1 and 6.69 mg TE.g-1 for the EA and BuOH fraction, respectively)

25 were much lower compared to those of the positive controls of BHT(49.64 mg TE.g-1), ascorbic acid (240.69 mg

26 TE.g-1)andĮ-tocopherol (71.00 mg TE.g-1). It is likely that there was no, or very little amounts of tocopherols

27 or/and ascorbic acid in the extract and fractions compared to other brown algae reported (Farvin & Jacobsen,

28 2013; Fayaz et al., 2005). From these findings, compared to reports by previous studies, it could be suggested

29 that the phenolic compounds in the EA and BuOH fractions of the alga H. banksii possess high antioxidant

30 capacity and phenolics play the main role in antioxidant activities of the extract. In addition, the positive

8 1 correlation between phenolics and antioxidant activities of the alga H. banksii was also observed and supported

2 by our previous studies (Dang et al. 2017b), and the relationship between phenolics and cytotoxic activity of the

3 extract will be discussed below.

4 Cytotoxic activities of the fractions against several cancer cell lines

5 Many marine algae derived phenolic compounds have been shown to possess the growth inhibitory activity of

6 several cancer cell lines (Li et al. 2011; Sithranga Boopathy and Kathiresan 2010). Table 1 reveals that the crude

7 extract and phlorotannin-enriched fractions showed cytotoxic activity across the panel of cell lines with various

8 efficacies (using the MTT assay). The EA fraction showed high inhibitory activity • 80%) in all cancer cell

9 lines tested, particularly of the glioblastoma (SJ-G2), lung (H460), skin (A431) and pancreas (MiaCaPa2) cells

10 showing complete inhibition. The BuOH fraction also exhibited impressive inhibitory activity • 70%) in almost

11 all cancer cell lines. For the normal cells, the crude extract was shown to have low toxicity against normal breast

12 cells (Breast- MCF10A; 26% inhibition), while high toxicity was observed for both the EA and BuOH fractions

13 (at 100 μg.mL-1) with inhibitory values of 98 and 78%, respectively. In comparison to two fractions above, the

14 inhibitory activity of the crude extract was significantly lower across the panel of cell lines (20-46%).

15 Correlations between phenolic-enriched extracts and cancer cell growth inhibition have been demonstrated by

16 several previous studies. The findings were supported by Yuan and Walsh (2006) who reported that cytotoxic

17 activity against a human cervical adenocarcinoma cell line (HeLa cells) of algal extracts (A red alga, dulse

18 (Palmaria palmata) and three kelp (Laminaria setchellii; Macrocystis integrifolia; Nereocystis leutkeana)) was

19 positive correlation to total phenolic content. The similar trend was observed in Sargassum muticum samples

20 against a human colon cancer cell line (HT-29) collected at different locations along the North Atlantic coasts

21 (Montero et al. 2016). The F3 fraction (the phenolic fraction) from Laurencia obusta exhibited significantly

22 higher cell growth inhibition against human tumor cell lines; A549 (lung cell carcinoma), HCT15 (colon cell

23 carcinoma) and MCF7 (breast adenocarcinoma) compared the crude extract and other fractions (Dellai et al.

24 2013). In regard to selective cytotoxicity, from the algal extract of Ecklonia cava, a CphF extract containing

25 high phenolic levels had high inhibitory activity on the murine colon cancer cell line (CT-26) and mouse

-1 26 melanoma cell line (B-16) with IC50 values of 5.1 and 29.3 μg.mL , respectively. Importantly, this extract

27 showed only slight toxicity to the normal cells (V79-4) with inhibition < 20% at 5-100 μg.mL-1 (Athukorala,

28 Kim, & Jeon, 2006). In addition, Khanavi et al. (2010) revealed that the hexane fraction from Sargassum

29 swartzii and Cystoseira myrica showed selective cytotoxicity against Caco-2 cells (colon adenocarcinoma; IC50

9 -1 -1 1 <100 μg.mL ), T47D cells (breast carcinoma; IC50 <100 μg.mL ), while it demonstrated low toxicity to the

2 normal cells NIH 3T3 (Swiss embryo fibroblast).

3 The anticancer activity of a number of marine phenolic compounds has been postulated through several different

4 mechanisms, including anti-proliferation, induction of apoptosis, cell cycle arrest and anti-angiogenesis,

5 immunomodulatory effects, antimitogenic activity, cell migration effects (Wu et al. 2016). Phlorotannins with

6 high radical scavenging capacity can protect human body against cancers due to the formation of cancer cells in

7 human body directly caused by free radicals (Lobo et al. 2010). In addition, anticancer effects of phlorotannins

8 was shown via enzyme inhibitory activity such as matrix metalloproteinase enzymes (MMPs) closely associated

9 with tumor invasion and metastasis in pathological conditions, while hyaluronidase enzyme is known to be

10 involved in allergic effects, migration of cancer and inflammation (Li et al. 2011). Moreover, it was reported

11 that phlorotannins (dieckol) induced apoptosis via the activation of both death receptor and mitochondrial-

12 dependent pathways in human hepatocellular carcinoma Hep3B cells (Yoon et al. 2013), and via ROS (reactive

13 oxygen species) production and the regulation of AKT and p38 signaling in ovarian cancer cells (Ahn et al.,

14 2015). Antiangiogenic activity of phlorotannins was also observed into fertilised chicken eggs through the

15 chorioallantoic membrane (CAM) (Namvar et al.2013).

16 Cytotoxic activities of the fractions against pancreatic cancer cell lines

17 The discrepancies in the percentage of cell growth inhibition against pancreatic cancer cell line (MiaCapa2) by

18 these phenolic fractions obtained from the MTT and CCK-8 assays (Table 1, 2) can be explained by the

19 differences in the mechanisms of the assays. The MTT assay measures only the mitochondrial dehydrogenase

20 activity of the cells, while most of the dehydrogenase activity in a cell was estimated in the CCK-8 assay

21 (Ishiyama et al. 1996). In addition, phenolic compounds (in the MTT assay) may affect the results by reducing

22 the tetrazolium salt even in the absence of cells (Maioli et al. 2009). However, the MTT assay was widely

23 applied to evaluate cell growth inhibition by the extracts across a panel of cancer cells as it is routinely used for

24 high-throughput drug screening with the strong correlation (r > 0.98) between the optical densities and cell

25 numbers (Hussain et al. 1993). For the CCK-8 assay, it has been demonstrated to be higher detection sensitivity

26 than the MTT, XTT and WST-8 assays (Kuhn et al. 2003; Jiao et al. 2015). Therefore, studies were extended to

27 assess the effects of the H. banksii fractions on four pancreatic cell lines (MiaPaCa2, BxPC3, CFPAC1 and

28 HPDE) using the CCK-8 assay (25-200 μg.mL-1).

29 In regard to activity against MiaPaCa2 cells, the EA fraction had high cell growth inhibition at almost all

30 concentrations (Table 2). There was no significant difference in the inhibitory values of the EA fraction at the

10 1 concentrations of 100-200 μg.mL-1 (P>0.05). The suppression of cancer cell growth by the EA fraction was

2 found to be efficacious (79.42%) even at the low concentration of 50 μg.mL-1, while it was only 33.23% with

3 the treatment of 25 μg.mL-1. No significant difference was found in the inhibition of cell growth by gemcitabine

4 (50nM) and the BuOH fraction of at the concentrations • 150 μg.mL-1 (P>0.05). For the BxPC3 cell line, no

5 significant cell inhibition was observed following treatment with the EA fraction at 100-200 μg.mL-1,andthe

6 EA fraction showed significantly higher inhibition than gemcitabine (75.03%) (P<0.05). The EA fraction still

7 exhibited high cell growth inhibition by 74.21% at concentration of 50 μg.mL-1. For the BuOH fraction, the high

8 cell growth suppression (> 70%) was observed at concentrations • 100 μg.mL-1, however, low inhibitory

9 efficacies (33.26 and 20.65%) were observed at the low concentrations (50 and 25 μg.mL-1), respectively. For

10 the secondary pancreatic cancer cell line (CFPAC1), the inhibitory efficacy of the EA fraction was high

11 (93.50%), even at the low dose of 50 μg.mL-1. The BuOH fraction was shown to be efficacious againstCFPAC1

12 FHOOVZLWKWKHWUHDWPHQWV•—JP/-1. There was no significant difference in the growth suppression between

13 the BuOH fraction (concentrations • μg.mL-1) and gemcitabine (50nM~13.16x10-3 μg.ml-1). It can be seen

14 that the phlorotannin-enriched fractions possessed high inhibitory activity against MiaPaCa2, BxPC3 and

15 CFPAC1 cells when compared to gemcitabine (50nM). These data warrant future investigations into identifying

16 the individual biologically active compounds contributing to this activity as well as their mechanisms of action.

17 In the HPDE cells, it was shown that the medium-polar phlorotannins in the EA fraction had the high toxicity to

-1 18 the normal cells (IC50 = 1.72 μg.mL ; Table 3). There were no significant differences in the toxicity of the EA

19 fraction at the concentrations of 200, 150 and 100 μg.mL-1, and gemcitabine (50nM) with the values of the

20 growth inhibition ~90% (P<0.05). The toxicity of this fraction was still high (inhibition >70%) when the cells

21 were treated with the low concentrations of 25-50 μg.mL-1. On the other hand, the toxicity of the BuOH fraction

-1 22 (the polar phlorotannins) was found to be low to the normal HPDE cells (IC50 = 106.45 μg.mL ; Table 3) with

23 61.30% of cells was suppressed at the high concentration of 150 μg.mL-1, while it was only 35.96% with the

24 dose of 100 μg.mL-1. It was interesting that at these concentrations, both the primary and secondary cancer cells

25 were inhibited by over 70%.

26 The findings were supported by McCauley et al. (2015) that the EA extracts from algal species; Hormosira

27 banksii, Solieria robusta and Ulvan sp. showed high cytotoxic activities (76-100%) against pancreatic cancer

28 (Miacapa-2). It notes that the Ulva sp. and H. banksii ethyl acetate extracts had selective cytotoxicity towards a

29 pancreatic cancer cell line with no toxicity towards a normal murine cell line at 100 μg.mL-1. Several pancreatic

30 cancer cell lines (Miacapa-2, Panc 3.27, Panc 1 and BxPC-3) were used to assess the activities of the organic

11 1 fraction of five brown algae; Dictyota dichotoma, Hormophysa triquerta, Spatoglossum asperum,

2 Stoechospermum marginatum and Padina tetrastromatica. Almost all polyphenol enriched fractions (the DCM

3 and EA fractions) of these algae showed high anti-proliferative activities (50–75%) against pancreatic cancer

4 and the apoptotic characteristics were observed via differential DNA fragmentation magnitudes in cancer cell

5 lines treated with these fractions (Aravindan et al.2013).

6 As discussed above, it is clear that phenolic compounds from brown alga H. banksii exhibited high cytotoxic

7 activity (almost all inhibition > 80%) against 11 cancer cell lines, while the crude extract was not efficacy for

8 this action (Table 1). In pancreatic cancer, high anticancer activity against three cell lines of phenolics was also

-1 -1 9 observed with the IC50 value < 30 μg.mL for the EA fraction and IC50 <65μg.mL for the BuOH fraction

10 (Table 3). From the findings and comparison to results reported in literature, it is suggested that phenolics from

11 brown alga H. banksii were the main contributors to cell growth inhibition on several cancer cell lines and high

12 efficacy against pancreatic cancer that had the limited therapeutic options. The individual phenolics from these

13 fractions will be further isolated and comprehensively assessed to elucidate their roles against pancreatic cancer.

14

15 Conclusions

16 Phlorotannin-enriched fractions were mainly responsible for antioxidant activities of the H. banksii extract and

17 exhibited high cytotoxic effects against several cancer cell lines. For pancreatic cancer cell lines, the

18 phlorotannin-enriched fractions showed excellent cytotoxic activities. The EA fraction possessed high cell

19 growth inhibition, even at low concentrations; however, it was toxic to the normal cells. The BuOH fraction

20 showed low toxicity to the normal cells, and was efficacious in the inhibition of the cancer cells. Therefore,

21 these phenolics will be further investigated for the effects on apoptotic and cell cycle mechanisms against

22 pancreatic cancer and as naturally biological compounds with potential applications in the pharmaceutical

23 industry.

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12 1 Acknowledgements

2 The authors also kindly thank to University of Newcastle; the Vietnamese Government through the Vietnam

3 International Education Development (VIED); the Ministry of Education and Training; the Ministry of

4 Agriculture and Rural Development for awarding a VIED-TUIT scholarship to Thanh Trung DANG.

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10 Conflict of interest

11 The authors declare no conflict of interest.

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13 1 References

2 Ahn JH, Yang YI, Lee KT, Choi JH (2015) Dieckol, isolated from the edible brown algae Ecklonia cava,

3 induces apoptosis of ovarian cancer cells and inhibits tumor xenograft growth. J Cancer Res Clin 141:255-

4 268

5 Aravindan S, Delma CR, Thirugnanasambandan SS, Herman TS, Aravindan N (2013) Anti-pancreatic cancer

6 deliverables from sea: first-hand evidence on the efficacy, molecular targets and mode of action for

7 multifarious polyphenols from five different brown-algae. PloS one 8:e61977

8 Athukorala Y, Kim KN, Jeon YJ (2006) Antiproliferative and antioxidant properties of an enzymatic

9 hydrolysate from brown alga, Ecklonia cava. Food Chem Toxicol44:1065-1074

10 Balboa EM, Conde E, Moure A, Falqué E, Domínguez H (2013) In vitro antioxidant properties of crude extracts

11 and compounds from brown algae. Food Chem138:1764-1785

12 Behrens D, Walther W, Fichtner I (2017) Pancreatic cancer models for translational research. Pharmacol

13 Therapeut 173:146-158

14 Benzie I, Strain J (1999) Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of

15 biological fluids and modified version for simultaneous measurement of total antioxidant power and

16 ascorbic acid concentration. Method Enzymol299:15-27

17 Bhuyan DJ, Vuong QV, Bond DR, Chalmers AC, Altena IA, Bowyer MC, Scarlett CJ (2017) Exploring the

18 least studied Australian eucalypt genera: Corymbia and Angophora for phytochemicals with anticancer

19 activity against pancreatic malignancies. Chem Biodivers 14:e1600291. doi: 10.1002/cbdv.201600291

20 Brand-Williams W, Cuvelier M, Berset C (1995) Use of a free radical method to evaluate antioxidant activity.

21 LWT-Food Sci Technol 28:25-30

22 Chakraborty K, Praveen NK, Vijayan KK, Rao GS (2013) Evaluation of phenolic contents and antioxidant

23 activities of brown seaweeds belonging to Turbinaria spp. (Phaeophyta, Sargassaceae) collected from Gulf

24 of Mannar. Asian Pac J Trop Biomed 3:8-16

25 Chandini SK, Ganesan P, Bhaskar N (2008) In vitro antioxidant activities of three selected brown seaweeds of

26 India. Food Chem 107:707-713

27 Cho M, Kang IJ, Won MH, Lee HS, You S (2010) The antioxidant properties of ethanol extracts and their

28 solvent-partitioned fractions from various green seaweeds. J Med Food 13:1232-1239

29 Cho M, Lee HS, Kang IJ, Won MH, You S (2011) Antioxidant properties of extract and fractions from

30 Enteromorpha prolifera, a type of green seaweed. Food Chem 127:999-1006

14 1 Chuen TL, Vuong QV, Hirun S, Bowyer MC, Predebon MJ, Goldsmith CD, Scarlett CJ (2016) Antioxidant and

2 anti-proliferative properties of Davidson’s plum (Davidsonia pruriens F. Muell) phenolic-enriched extracts

3 as affected by different extraction solvents. J Herb Med6:187-192

4 Dang TT, Vuong QV, Schreider MJ, Bowyer MC, Van Altena IA, Scarlett CJ (2017a). Optimisation of

5 ultrasound-assisted extraction conditions for phenolic content and antioxidant activities of the alga

6 Hormosira banksii using response surface methodology. J Appl Phycol 29: 3161-3173

7 Dang TT, Vuong QV, Schreider MJ, Bowyer MC, Van Altena IA, Scarlett CJ (2017b) The Effects of Drying on

8 Physico-Chemical Properties and Antioxidant Capacity of the Brown Alga (Hormosira banksii (Turner)

9 Decaisne). J Food Process Pres 41:e13025.doi:10.1111/jfpp.13025

10 Dellai A, Laajili S, Le Morvan V, Robert J, Bouraoui A. (2013). Antiproliferative activity and phenolics of the

11 Mediterranean seaweed Laurencia obusta. Ind Crop Prod 47:252-255

12 Duan XJ, Zhang WW, Li XM, Wang BG (2006) Evaluation of antioxidant property of extract and fractions

13 obtained from a red alga, Polysiphonia urceolata. Food Chem95:37-43.

14 El Gamal AA (2010) Biological importance of marine algae. Saudi Pharm J 18:1-25

15 Eom SH, Kim YM, Kim SK (2012) Antimicrobial effect of phlorotannins from marine brown algae. FoodChem

16 Toxicol 50:3251-3255

17 Farvin KS, Jacobsen C (2013) Phenolic compounds and antioxidant activities of selected species of seaweeds

18 from Danish coast. Food Chem 138:1670-1681

19 Fayaz M, Namitha K, Murthy KC, Swamy MM, Sarada R, Khanam S, Ravishankar G (2005) Chemical

20 composition, iron bioavailability, and antioxidant activity of Kappaphycus alvarezzi (Doty). J Agri Food

21 Chem 53:792-797

22 Ganesan P, Kumar CS, Bhaskar N (2008) Antioxidant properties of methanol extract and its solvent fractions

23 obtained from selected Indian red seaweeds. Bioresource Technol 99:2717-2723

24 Grasso C, Jansen G, Giovannetti E (2017) Drug resistance in pancreatic cancer: Impact of altered energy

25 metabolism. Crit Rev Oncol/Hematol114:139-152

26 Gupta S, Abu-Ghannam N (2011) Bioactive potential and possible health effects of edible brown seaweeds.

27 Trends Food Sci Technol 22:315-326

28 Hussain R, Nouri A, Oliver R (1993) A new approach for measurement of cytotoxicity using colorimetric assay.

29 J Immunol Methods 160: 89-96

15 1 Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno K (1996) A combined assay of cell vability

2 and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol

3 Pharma Bull 19: 1518-1520

4 Kang SM, Kim AD, Heo SJ, Kim KN, Lee SH, Ko SC, Jeon YJ (2012) Induction of apoptosis by

5 diphlorethohydroxycarmalol isolated from brown alga, Ishige okamurae. J Funct Food 4: 433-439

6 Kang YM, Eom SH, Kim YM (2013) Protective effect of phlorotannins from Eisenia bicyclis against

7 lipopolysaccharide-stimulated inflammation in HepG2 cells. Environ Toxicol Phar 35: 395-401

8 Khanavi M, Nabavi M, Sadati N, Shams Ardekani M, Sohrabipour J, Nabavi SMB, Ostad SN (2010) Cytotoxic

9 activity of some marine brown algae against cancer cell lines. Biol Res 43:31-37

10 Kubanek J, Lester SE, Fenical W, Hay ME (2004) Ambiguous role of phlorotannins as chemical defenses in the

11 brown alga Fucus vesiculosus. Mar Ecol Prog Ser277:79-93

12 Kuhn D, Balkis M, Chandra J, Mukherjee P, Ghannoum M (2003) Uses and limitations of the XTT assay in

13 studies of Candida growth and metabolism. J Clin Microbiol 41:506-508

14 Lee MS, Shin T, Utsuki T, Choi JS, Byun DS, Kim HR (2012) Isolation and identification of phlorotannins

15 from Ecklonia stolonifera with antioxidant and hepatoprotective properties in tacrine-treated HepG2 cells.

16 J Agric Food Chem 60: 5340-5349

17 Li YX, Wijesekara I, Li Y, Kim SK (2011) Phlorotannins as bioactive agents from brown algae. Process

18 Biochem 46:2219-2224

19 Lobo V, Patil A, Phatak A, Chandra N (2010) Free radicals, antioxidants and functional foods: Impact on human

20 health. Pharmacogn Rev 4: 118

21 Maioli E, Torricelli C, Fortino V, Carlucci F, Tommassini V, Pacini A (2009) Critical appraisal of the MTT

22 assay in the presence of rottlerin and uncouplers. Biol Proced online 11:227

23 Martínez JHI, Castañeda HGT (2013) Preparation and chromatographic analysis of phlorotannins. J Chromatogr

24 Sci 51:825-838

25 McCauley JI, Meyer BJ, Winberg PC, Ranson M, Skropeta D (2015) Selecting Australian marine macroalgae

26 based on the fatty acid composition and anti-inflammatory activity. J Appl Phycol 27: 2111-2121

27 Montero L, Sánchez-Camargo AP, García-Cañas V, Tanniou A, Stiger-Pouvreau V, Russo M, Ibáñez E (2016)

28 Anti-proliferative activity and chemical characterization by comprehensive two-dimensional liquid

29 chromatography coupled to mass spectrometry of phlorotannins from the brown macroalga Sargassum

30 muticum collected on North-Atlantic coasts. J Chromatogr A1428:115-125

16 1 Namvar F, Mohamad R, Baharara J, Zafar-Balanejad S, Fargahi F, Rahman HS (2013) Antioxidant,

2 antiproliferative, and antiangiogenesis effects of polyphenol-rich seaweed (Sargassum muticum). BioMed

3 Res Inter 2013. doi.org/10.1155/2013/604787

4 Raviv NV, Sakhuja S, Schlachter M, Akinyemiju T (2017) Metabolic syndrome and in-hospital outcomes

5 among pancreatic cancer patients. Diabetes Metab Syndr: Clin Res Rev (In Press).

6 doi.org/10.1016/j.dsx.2017.04.019

7 Scarlett CJ, Salisbury EL, Biankin AV, Kench J (2011) Precursor lesions in pancreatic cancer: morphological

8 and molecular pathology. Pathol 43:183-200

9 Schumacher M, Kelkel M, Dicato M, Diederich M (2011) Gold from the sea: marine compounds as inhibitorsof

10 the hallmarks of cancer. Biotechnol Adv29:531-547

11 Shibata T, Yamaguchi K, Nagayama K, Kawaguchi S, Nakamura T (2002) Inhibitory activity of brown algal

12 phlorotannins against glycosidases from the viscera of the turban shell Turbo cornutus. European J Phycol

13 37:493-500

14 Singh IP, Bharate SB (2006) Phloroglucinol compounds of natural origin. Nat Prod Rep 23:558-591

15 Sithranga Boopathy N, Kathiresan K (2010) Anticancer drugs from marine flora: an overview. J Oncology

16 2010. doi.org/10.1155/2010/214186

17 6NHUJHW0.RWQLN3+DGROLQ0+UDV$56LPRQL0.QH]=  3KHQROVSURDQWKRF\DQLGLQVÀDYRQHV and

18 Àavonols in some plant materials and their antioxidant activities. Food Chem 89:191-198

19 Thaipong K, Boonprakob U, Crosby K, Cisneros-Zevallos L, Hawkins Byrne D (2006) Comparison of ABTS,

20 DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. J Food

21 Compo Anal 19:669-675

22 Wang BG, Zhang WW, Duan XJ, Li XM (2009a) In vitro antioxidative activities of extract and semi-purified

23 fractions of the marine red alga, Rhodomela confervoides (Rhodomelaceae). Food Chem 113:1101-1105

24 Wang T, Jonsdottir R, Ólafsdóttir G (2009b) Total phenolic compounds, radical scavenging and metal chelation

25 of extracts from Icelandic seaweeds. Food Chem116:240-248

26 Wang T, Jónsdóttir RS, Liu H, Gu L, Kristinsson HG, Raghavan S, Ólafsdóttir GN (2012) Antioxidant

27 capacities of phlorotannins extracted from the brown algae Fucus vesiculosus. J Agri Food Chem 60:5874-

28 5883

29 Wu L, Sun J, Su X, Yu Q, Yu Q, Zhang P (2016) A review about the development of fucoidan in antitumor

30 activity: Progress and challenges. Carbohyd Polym 154:96-111

17 1 Jiao G, He X, Li X, Qiu J, Xu H, Zhang N, Liu S (2015) Limitations of MTT and CCK-8 assay for evaluation of

2 graphene cytotoxicity. RSC Adv 5: 53240-53244

3 Yoon JS, Yadunandam AK, Kim SJ, Woo HC, Kim HR, Kim GD (2013) Dieckol, isolated from Ecklonia

4 stolonifera, induces apoptosis in human hepatocellular carcinoma Hep3B cells. J Nat Med 67: 519-527

5 Yuan YV, Walsh NA (2006) Antioxidant and antiproliferative activities of extracts from a variety of edible

6 seaweeds. Food Chem Toxicol 44:144-1150

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18 1 Figure Capture

2 Figure 1. TLC for the H. banksii fractions; (F3 - ethyl acetate (EA) fraction; F4 – butanol 3 (BuOH) fraction; and F5 – aqueous fraction). The bands were visualised by using a vanillin 4 stain

5 Figure 2. Total phenolic content (TPC) of the crude extract and phlorotannin-enriched 6 fractions 7

8 Figure 3. Antioxidant activities of the extract and phlorotannin-enriched fractions compared 9 to the positive controls (butylated hydroxytoluene (BHT); ascorbic acid (A.A) and Į- 10 WRFRSKHURO Į-Toco) at a concentration of 0.12 mg.mL-1

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19 Table 1. Cell growth inhibition (%) screened across various cancer cell lines in response to the crude extract, the EA and BuOH fraction (100 μg.mL-1) using the MTT assay Cell line Cancer cell type Crude extract EA fraction BuOH fraction HT29 Colon 22±6 90±4 77±4 U87 Glioblastoma 22±5 98±3 71±4 SJ-G2 Glioblastoma 20±2 >100 >100 SMA Glioblastoma (Murine) 52±19 95±3 94±1 MCF-7 Breast 30±11 81±3 69±6 A2780 Ovarian 37±9 87±1 81±3 H460 Lung 18±5 >100 >100 A431 Skin 32±4 >100 >100 Du145 Prostate 23±8 80±6 38±4 BE2-C Neuroblastoma 49±11 97±4 88±3 MiaCaPa2 Pancreas 46±16 >100 98±2 MCF10A Breast (Normal) 26±10 98±2 78±3 Higher values indicate greater inhibition. The values are the mean ± standard deviation for at least triplicates (n=3-6).

Table 2. The cytotoxic activities of the phlorotannin-enriched fractions (EA, BuOH fraction) at different concentrations compared to gemcitabine (50nM) in pancreatic cell lines (the CCK-8 assay) Concentration of the fraction (μg.mL-1) and gemcitabine (50 nM) Cell Lines Fractions 200 150 100 50 25 Gem. a a a b c EA 89.46±2.11 88.48±2.04 87.72±1.95 79.76±1.05 71.95±1.25 a HDPE b c d e BuOH 80.84±3.91 61.3±3.86 35.96±2.50 19.72±2.81 f 89.67±0.98 a a a b 9 11±4 1 c EA 93.35±0.45 93.43±0.43 93.59±1.14 79.42±3.33 33.23±2.51 d MiaCaPa2 d d b c BuOH 91.08±0.51 90.58±3.41 79.14±3.14 32.53±2.08 e 87.28±1.59 a a a b 22 49±3 12c EA 94.19±1.51 94.25±1.40 94.65±1.14 74.21±2.62 40.65±2.79 b BxPC3 a a b c BuOH 86.72±2.34 85.77±1.89 73.45±3.94 33.26±2.13 d 75.03±1.28 a a a a 20 65±1 87b EA 94.21±1.39 94.39±0.33 94.35±2.33 93.5±4.98 59.09±2.16 a CFPAC1 93.05±2.42a 90.82±2.49a 79.02±3.08b 46.86±1.78c 92.42±2.34 BuOH 30 78±3 21d All values are means ± standard deviation and those in the same row not sharing the same superscript letter are significantly different from the others (P<0.05). -1 Table 3. IC50 values (μg.mL ) of the phlorotannin-enriched fractions from H.banksii on pancreatic cell lines IC Value (μg.mL-1) Cell lines 50 EA fraction BuOH fraction HDPE 1.72 106.45 MiaCaPa2 29.08 58.06 BxPC3 25.17 62.18 CFPAC1 6.71 47.12 )LJXUH &OLFNKHUHWRGRZQORDG)LJXUH)LJXUHBWLII )LJXUH &OLFNKHUHWRGRZQORDG)LJXUH)LJXUHGRF[ )LJXUH &OLFNKHUHWRGRZQORDG)LJXUH)LJXUHGRF[ Paper VIII:

“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.

Manuscript Details

Manuscript number BIOPHA_2018_2938

Title Extraction and cytotoxic activity of polysaccharides (fucoidans) against pancreatic cancer in vitro from brown alga Hormosira banksii (Turner) Decaisne

Article type Research Paper

Abstract Brown algae are a rich source of bioactive compounds, especially polysaccharides that have potential for the treatment of several cancers. This study aimed to evaluate the antioxidant and cytotoxic activities of the polysaccharide fractions (three fractions; CF50, CF70 and CFR) from the brown algae Hormosira banksii on pancreatic cancer cell lines (MiaCaPa2, BxPC3 and CFPAC1) as well as non-tumorigenic cells (HPDE). The results revealed that sulfated groups were observed in all polysaccharide fractions. Antioxidant activities (the ABTS, DPPH and FRAP assays) of these polysaccharides were low. However, all these fractions possessed high cytotoxic activity against pancreatic cancer cell lines (MiaCaPa2, BxPC3 and CFPAC1) in a dose-dependent manner, while the CF50 and CF70 fractions were low toxicity towards normal cells. The findings showed potential of polysaccharides from the H.banksii extract against pancreatic cancer and further investigations should be conducted for delineation of their effects on apoptotic and cell cycle mechanisms.

Keywords Sulfated polysaccharides (Fucoidans); Brown algae; Cytotoxic activity; Pancreatic cancer.

Manuscript region of origin Asia Pacific

Corresponding Author Thanh Dang

Order of Authors Thanh Dang, Christopher Scarlett

Suggested reviewers Quanbin Zhang, SangGuan You, Hugo Alexandre Rocha

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Highlights.doc [Highlights]

Submission for Fucoidan (30.4.2018).docx [Manuscript File]

Tables for Fucoidan.docx [Table]

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Tuesday 1th May, 2018

Prof. D.M. Townsend, and Prof. L.E. Wold Editor-in-chief Journal of Biomedicine and Pharmacotherapy

Dear Prof. D.M. Townsend, and Prof. L.E. Wold,

On behalf of my co-authors, I would like to submit our original manuscript entitled “Extraction and cytotoxic activity of polysaccharides (fucoidans) against pancreatic cancer in vitro from brown alga Hormosira banksii (Turner) Decaisne”, which we wish to be considered for publication as an original research article in Journal of Biomedicine and Pharmacotherapy.

Hormosira banksii is a brown fucoid alga rich in bioactive compounds, and is widely distributed in the intertidal areas along the eastern coast of Australia. This study aimed to evaluate the antioxidant and cytotoxic activity of the polysaccharide fractions from the brown algae Hormosira banksii on pancreatic cancer cell lines (MiaCaPa2, BxPC3 and CFPAC1) as well normal pancreatic epithelial cells (HPDE). The results revealed that all the polysaccharide fractions (CF50, CF70 and CFR) were found to be presence of sulphate content (these polysaccharides called fucoidans). Antioxidant activities (the ABTS, DPPH and FRAP assays) of these sulphate polysaccharide were weak. However, these fractions were shown to be high cytotoxic activities against pancreatic cancer cell lines (MiaCaPa2, BxPC3 and CFPAC1) in a dose-dependent manner. In addition, the CF50 and CF70 fractions were low toxicity to the non- tumourigenic cells (HPDE). The findings showed potentials of polysaccharides from the H.banksii extract against pancreatic cancer and further investigations should be conducted for delineation of their effects on apoptotic and cell cycle mechanisms.

Our manuscript reports previously unpublished work and has not been submitted simultaneously, in whole or in part, to another journal. The manuscript has been seen and approved by all authors and have agreed to submit this manuscript to Journal of Biomedicine and Pharmacotherapy in its present form. We hope that you and the reviewers share our enthusiasm for these data, and we look forward to receiving your comments in due course.

With my best regards, A/Prof. Christopher Scarlett, PhD

Deputy Head of School (Ourimbah) Head Discipline of Applied Science, School of Environmental and Life Sciences. Head, Pancreatic Cancer Research, University of Newcastle. 10 Chittaway Rd, Ourimbah, NSW 2258, Australia. Tel.: +61 2 4348 4680; Fax: +61 243484145; E-mail: [email protected] Highlights

- Sulfate content was found in all three polysaccharide fractions from Hormosira banksii.

- Sulfate content and molecular weight affected antioxidant and cytotoxic activities of the polysaccharide fractions.

- These polysaccharide fractions exhibited high activity against pancreatic cancer cell lines, while low toxicity towards the non-tumorigenic cells. 1 Extraction and cytotoxic activity of polysaccharides (fucoidans) against pancreatic 2 cancer in vitro from brown alga Hormosira banksii (Turner) Decaisne

3

4 Thanh T. Dangab, Christopher J. Scarletta*

5

6 a School of Environmental and Life Sciences, Faculty of Science, University of Newcastle, Ourimbah, New South Wales, 7 Australia

8 b Department of Seafood Processing Technology, Faculty of Food Technology, Nha Trang University, Nha Trang, Khanh Hoa, 9 Vietnam

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15 Running Title: Sulfated polysaccharides and anticancer activity of the H. banksia extract.

16

17 *Corresponding author:

18 A/Prof. Christopher J. Scarlett, PhD

19 Deputy Head of School (Ourimbah). 20 Head Discipline of Applied Science, 21 School of Environmental and Life Sciences. 22 Head, Pancreatic Cancer Research, 23 University of Newcastle. 24 10 Chittaway Rd, Ourimbah, NSW 2258, Australia. 25 26 Ph: +61 2 4348 4680 Fax: +61 2 4348 4145 27 Email: [email protected]

28 1 Abstract

2 Brown algae are a rich source of bioactive compounds, especially polysaccharides that have

3 potential for the treatment of several cancers. This study aimed to evaluate the antioxidant and

4 cytotoxic activities of the polysaccharide fractions (three fractions; CF50, CF70 and CFR) from

5 the brown algae Hormosira banksii on pancreatic cancer cell lines (MiaCaPa2, BxPC3 and

6 CFPAC1) as well as non-tumorigenic cells (HPDE). The results revealed that sulfated groups

7 were observed in all polysaccharide fractions. Antioxidant activities (the ABTS, DPPH and

8 FRAP assays) of these polysaccharides were low. However, all these fractions possessed high

9 cytotoxic activity against pancreatic cancer cell lines (MiaCaPa2, BxPC3 and CFPAC1) in a

10 dose-dependent manner, while the CF50 and CF70 fractions were low toxicity towards normal

11 cells. The findings showed potential of polysaccharides from the H.banksii extract against

12 pancreatic cancer and further investigations should be conducted for delineation of their effects

13 on apoptotic and cell cycle mechanisms.

14

15 Keyword: Sulfated polysaccharides (Fucoidans); Brown algae; Cytotoxic activity; Pancreatic

16 cancer.

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22 1 1. Introduction

2 Marine algae are widely used in traditional food and as the source of bioactive compounds with

3 diverse structures and biological activities applied to develop new drugs [1]. The algae contain

4 the polysaccharides such as alginates, carrageenans, laminarans, agars and sulfated

5 polysaccharides (fucoidans). The red algae are known to produce carrageenans and agars, while

6 alginates, laminarin and sulfated polysaccharides are mainly obtained from brown algae [2, 3]. In

7 particular, fucoidans are increasingly attracted in the search for the pharmaceutical applications

8 [4]. Fucoidan is a cell wall sulfated polysaccharide comprised of fucose, uronic acids, galactose,

9 mannose, glucuronic acid, xylose and sulfate group that exhibits a diversity of biological

10 activities, including anti-inflammatory, antioxidant, antiviral, anticoagulant/thrombotic and

11 anticancer [5]. The molecular weight of fucoidan polymers widely ranged depended on the algal

12 species, environmental conditions and extraction methods. It is generally classified into three

13 types as low (< 10 kDa), medium (10 kDa - 10.000 kDa) and high (> 10.000 kDa) [6]. Low

14 molecular weight fucoidans have been demonstrated to enhance their biological activities

15 compared to native fucoidans [7]. The changes in the molecular weight of fucoidan polymers,

16 number and position of sulfate groups, and monosaccharide composition also affect their

17 biological activities [8]. Fucoidan could be extracted from brown algae using acid solution or

18 water. There was no standardised purification procedure available for polysaccharides. In almost

19 all methods, pure ethanol was used to remove residual components (pigments, lipids,

20 proteins…), and to precipitate crude polysaccharides or create polysaccharide fractions, while

21 calcium cation (Ca2+) solution could be used to promote alginate precipitation during a

22 purification process [4]. 1 Pancreatic cancer is the leading cause of cancer-related death in the Western countries and in the

2 world due to the late onset of symptoms and diagnosis at advanced stage [9]. The high mortality

3 rate is also due to the lack of effective treatments. Pancreatic ductal adenocarcinoma (PDAC) is

4 the most common cancer type and surgical resection was applied for a small proportion (15-

5 20%) of patients at early stage for prolonging survival with only a few months [10]. Gemcitabine

6 (2-2-difluorodeoxycytidine) have been a first line therapy for the treatment of pancreatic cancer

7 for several decades. The combination of gemcitabine with numerous agents (5-fluorouracil,

8 cisplatin, oxaliplatin, epirubicin, irinotecan, capecitabine, docetaxel, and mitomycin C) have

9 been estimated in order to improve the therapeutic efficacies, but no clear benefits for overall

10 survival were observed, while these agents were found to be toxic to the normal cells [11]. The

11 biologically active components within the algal extracts are considered as an adjuvant therapy

12 for certain cancers. The active algal polysaccharides are particularly important for the treatment

13 of several cancers with very limited therapeutic options such as pancreatic cancer [12].

14 Till now, there is no assessment of the polysaccharides from brown alga Hormosira banksii for

15 the treatment of pancreatic cancer reported. The aims of our work were to isolate the sulfated

16 polysaccharides from this source. The antioxidant and cytotoxic activity of these polysaccharides

17 against pancreatic cancer were evaluated via in vitro tests and further applications in food and

18 pharmaceutical fields.

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22 1 2. Materials and methods

2 2.1 Materials and sample preparation

3 The brown alga Hormosira banksii was collected in March, 2016 from the rocky shore at Bateau

4 Bay, NSW, Australia (Latitude of 33°22'55.2"S; longitude of 151°29'6"E). After collection, the

5 sample was washed with seawater to remove residues (sand and epifauna). At the laboratory, the

6 alga was washed thoroughly with freshwater and freeze dried for 48 h using a freeze dryer

7 (Thomas Australia Pvt. Ltd., Seven Hills, NSW, Australia). The dried samples were pulverized,

8 sieved (” 600 μm particle size) and stored at – 20 °C for further analysis.

9 2.2 Extraction of the polysaccharides

10 The procedure was conducted as described by previous studies [8, 13] with slight modifications.

11 Briefly, the fine algal powder (10 g) was treated with the solvent ethanol 99.5% (300 mL) and

12 stirred using a magnetic bar for 24 hours at room temperature. The supernatant (pigments, lipids

13 and proteins) was discarded by centrifugation (3000 rpm, for 10 min at 4 °C) and the process

14 was repeated till the samples became colourless (n=3). The biomass was continuously extracted

15 by water using ultrasonic bath (Soniclean, 220 V, 50 Hz and 250W, Soniclean Pty Ltd,

16 Australia) set at temperature of 30 °C, time of 60 min and power of 150W as described by Dang

17 et al. [14]. The extracts were centrifuged (5000 rpm, for 20 min at 4 °C) to remove the residues.

18 The process was repeated (n=5) till almost all polysaccharides were isolated. The crude

19 polysaccharides were mixed with 1% CaCl2 and kept at 4 °C overnight to precipitate alginic acid.

20 The supernatant was collected by centrifugation (5000 rpm, for 20 min at 4 °C) and the crude

21 polysaccharides were added with ethanol (99.5%) to obtain the final ethanol concentrations of

22 50%, 70%. The fractions of ethanol 50% (CF50), ethanol 70% (CF70) and residue (CFR) were

23 concentrated and freeze dried to the powders and stored at – 20 °C for further analysis. 1 2.3 Determination of sulfate content and total sugars

2 The sulfate content of the polysaccharide fractions was determined by the BaCl2-gelatin method

3 [15, 16] using K2SO4 as a standard after hydrolysing polysaccharides in 4 M HCl for 2 h at 100

4 °C. Briefly, 0.2 mL sample in the tube was added 3.8 ml trichloro-acetic acid 4%, and then 1 ml

° 5 BaCl2-gelatin (2g gelation dissolved in 400 ml water 70 C and mixed with 2 g BaCl2). The

6 sample was well mixed for 20 min and measured at 360 nm using an UV-vis spectrophotometer

7 (Varian Australia Pty. Ltd., Victoria, Australia). The content of total sugars was evaluated by the

8 phenol–sulfuric acid method [17, 18] using D-glucose as a standard. Briefly, 0.5 mL sample was

9 added with 0.5 mL of phenol 5% (w/v) and 2.5 mL of sulphuric acid 98%. The samples were

10 vortexed and kept for 20 min at room temperature; and the absorbance of the sample was

11 measured at 490 nm.

12 2.4 Total phenolic content (TPC) of the fractions

13 Total phenolic content (TPC) of the polysaccharide fractions was determined as described by

14 Dang et al. [14] using the Folin-Ciocalteu method. The absorbance was measured at 765 nm.

15 Gallic acid was used as the standard and the results were expressed as mg of gallic acid

16 equivalents per gram of the sample (mg GAE.g-1).

17 2.5 Antioxidant activities of the fractions

18 ABTS total antioxidant capacity (ABTS) was measured as described by Thaipong et al. [19].

19 Methanol and trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid) were used as a

20 control and a standard, respectively. The absorbance was measured at 734 nm and the results

21 were expressed as mg of trolox equivalents per gram of the sample (mg TE.g-1). 1 DPPH Free radical scavenging capacity (DPPH): The extract was analysed as described by

2 Brand-Williams et al. [20]. Trolox was used as the standard and the sample was read at 515nm.

3 The results were expressed as mg TE.g-1 the sample.

4 Ferric reducing antioxidant power (FRAP): The extract was carried out as described by Benzie

5 and Strain [21]. Trolox was used as a standard and sample was measured at 593nm. The results

6 were expressed as mg TE.g-1 the sample.

7 2.6 Determination of the cytotoxic activity of the fractions

8 Cell culture: Human pancreatic cancer cells (two primary pancreatic cancer cell lines:

9 MiaPaCa2, BxPC3 and one secondary pancreatic cancer cell line: CFPAC1) and Human

10 pancreatic ductal epithelial cells (HPDE) were cultured at 37 °C, 5% CO2. Keratinocyte Serum-

11 free Media (K-SFM) with human Recombinant Epidermal Growth Factor (EGF) and Bovine

12 Pituitary Extract (BPE) (10%) was used for HPDE. Dulbecco’s Modified Eagle’s Medium

13 (DMEM) supplemented with 10% foetal bovine serum (FBS), 2.5% horse serum and L-

14 glutamine (100 μg.mL-1) was used to culture MiaPaCa2 cells, while RPMI media with 10% FBS

15 and L-glutamine (100 μg.mL-1) was applied for BxPC3 and CFPAC1 cells were cultured in

16 IMDM media supplemented with 10% FBS and L-glutamine (100 μg.mL-1).

17 Cytotoxic activity of the fractions: Cytotoxic activity of polysaccharides was determined using

18 the Dojindo Cell Counting Kit-8 (CCK-8: Dojindo Molecular Technologies, Inc., Rockville,

19 MD, USA) assay. Pancreatic cells were seeded into a 96-well plate at 3x103 cells per well (200

20 μL) for MiaCaPa2 and 7x103 cells for HPDE, BxPC3 and CFPAC1, and incubated at 37 °C with

-1 21 5% CO2 for 24 h. The cells were then treated with various concentrations (25-200 μg.mL ) of

22 polysaccharides, gemcitabine (50 nM) and 0.5% DMSO as vehicle control. After 72 h, 10 μL of

23 CCK-8 solution was added before incubating at 37 °C with 5% CO2 for 90 min. The absorbance 1 was measured at 450 nm on a multi-well spectrophotometer (BIORAD Benchmark PlusTM), and

2 the cytotoxic activity of polysaccharides was determined as a percentage of dead cells compared

3 to control (DMSO) (% inhibition = ((Abs of the control – Abs of the sample)/Abs of the control

4 x 100%). Six repeats were performed for each concentration (n=6).

5 2.7 Statistical Analysis

6 A one-way ANOVA and LSD post-hoc test were employed (SPSS Statistical Software, Version

7 16) to analyse the differences between the independent samples. Differences between the mean

8 levels of analyses were taken to be statistically significant at P<0.05. All experiments were

9 conducted at least in triplicate and the results were performed as means ± standard deviations.

10 The IC50 (the concentration required inhibits cell growth by 50%) values were calculated by

11 curve fitting the absorbance (viability) vs. log [concentration of treatment] using GraphPad Prism

12 software (version 7.03).

13 3. Results and Discussion

14 3.1 Compositional analysis

15 The recovery yields of the polysaccharides fractions from H. banksii are shown in Table 1. The

16 results showed that the yield of the CF50 fraction was the highest of 30.53% (% dry weight). The

17 CFR fraction was found to be 9.56%, while the lowest yield was for CF70 with 5.82%. In

18 comparison with the previous studies, the maximum yield of fucoidan obtained from Saccharina

19 japonica was 13.56% using subcritical water extraction [22]. The fucoidan yield from the alga

20 Undaria pinnatifida significantly changed from July (25.4–26.3%) to September (57.3–69.9%)

21 [16]. On the other hand, the low yield of polysaccharide fractions (0.30-0.96%) was also

22 observed from the Sargassum pallidum extract [23]. The considerable changes in the yields of

23 polysaccharides might be due to algal species, seasons, structure of materials and extraction 1 methods. Moreover, polysaccharides were usually separated by centrifugation and precipitation

2 [13, 18], while some polysaccharides were required to filter using a 0.45 μm filter before

3 precipitated in other approaches [8, 24]. This also resulted in significant differences in the yields

4 of polysaccharides.

5 Total phenolic content for the polysaccharide fractions was evaluated (Table 1). The highest TPC

6 value was of 90.77 mg GAE.g-1 for the CFR fraction at a concentration of 1.0 mg.mL-1. There

7 was no significant difference in total phenolics between the CF50 and CF70 fractions with 25.59

8 and 26.79 mg GAE.g-1, respectively. The results indicated that a high proportion of phenolics

9 was removed from the polysaccharide fractions compared with total phenolics of the crude

10 H.banksii extract (158.82 mg GAE.g-1) in our previous study [25]. The low ratio of phenolics in

11 the polysaccharide fractions was reported in previous studies. The phenolic content of the crude

12 polysaccharide fraction (CpoF; 241 μg/100 ml) from Ecklonia cava was lower than that of the

13 crude extract (OE; 301μg/100 ml) and the crude polyphenol fraction (CphF, 901 μg/100 ml)

14 [13]; and the TPC value was of 36.9 μg GAE.g-1 from the Sargassum binderi fucoidan extract

15 [26]. It is suggested that the polysaccharides could be more purity via a procedure (the

16 polysaccharides dissolved in water and then precipitated with pure ethanol) repeated several

17 times to remove the residues as ethanol-dissolved phenolics, pigments and others.

18 Total sugar and sulfate content of the polysaccharides fractions were evaluated using the phenol-

19 sulfuric acid and BaCl2–gelatine methods, respectively (Table 1). With the sugar content, the

20 CF50 fraction possessed the highest value with 29.31%, followed by the CF70 fraction with

21 23.21%, while the lowest value was for the CFR fraction with only 7.37%. It can be seen that

22 ethanol in the solution helps separate the polysaccharides with high molecular weight by

23 precipitating them (in the CF50 and CF70 fractions), while low weight polysaccharides not 1 precipitated at ethanol 70% and phenolics remained in the residue fraction (CFR). Therefore, a

2 low sugar ratio was observed in the residue fraction. There was a wide range of carbohydrates

3 (11.5–66%) in brown algae depending on species, growing conditions, extraction procedures and

4 analytical methods [8]. In addition, the different polysaccharides could be separated by various

5 ethanol percentages in the solution. Alginate sodium and fucoidan could be sequentially

6 precipitated by ethanol 20% and 50% in the solution, while the ratio of ethanol over 65% was

7 used to isolate laminaran [27]. However, from the findings, sulfated polysaccharides were

8 observed in all fractions. No significant differences in the sulfate content were found between the

9 CF50 and CFR fractions; and the CF70 and CFR fractions. The highest sulfate ratio was of

10 12.19% in the CF70 fraction and these values for the CF50 and CFR fraction were 10.59 and

11 11.00%, respectively. The sulfate ratio significantly affected biological activity of the

12 polysaccharides. The polysaccharide fractions from Porphyra haitanesis with high sulfate

13 content exhibited higher antioxidant activities compared to others [28], while there was a

14 positive correlation between sulfate content and inhibition of HeLa cell proliferation found from

15 the algae Caulerpa prolifera, Sargassum filipendula, Dictyopteris delicatula and Dictyota

16 menstruallis [29].

17 3.2. Antioxidant activity of the fractions

18 Three assays (ABTS, DPPH and FRAP) were used to evaluate antioxidant activities of the

19 polysaccharide fractions at a concentration of 1.0 mg.mL-1. The results were all presented in

20 Table 2. There were significant differences in antioxidant activities among the polysaccharide

21 fractions. Total antioxidant capacity (ABTS) based on the scavenging ability of antioxidants to

22 the radical anion ABTS*+ [19]. The significantly higher ABTS activity of the CFR fraction

23 (106.99 mg TE.g-1) was shown compared to these of the CF70 (17.49 mg TE.g-1) and CF50 1 (12.40 mg TE.g-1). It is could be that the phenolic content mainly affected ABTS activity of the

2 fractions. On the other hand, with no significant difference in the TPC values of CF50 and CF70,

3 higher ABTS activity in the CF70 than the CF50 could be due to higher sulfate content and

4 lower molecular weight of polysaccharides within the CF70 fraction. DPPH radical scavenging

5 capacity based on ability of a compound to scavenge DPPH radicals is dependent on their ability

6 to pair with the unpaired electron of a radical [20]. The similar trend was also found in the DPPH

7 assay with the highest value (103.05 mg TE.g-1) for the CFR fraction, while they were low for

8 the CF50 (8.66 mg TE.g-1) and CF70 (15.29 mg TE.g-1) fractions. It was supported by Mak et al.

9 [16] who indicated that the differences in DPPH radical scavenging activity were observed in the

10 polysaccharide fractions (made by ion-exchange chromatography) from Undaria pinnatifida; the

11 F3 (68.65%), F2 (58.65%) and F1 (53.45%) fractions, and the higher DPPH value was also

12 found in the F3 fraction related to its high sulfate content and molecular weight. For FRAP

13 assay, it based on the ability of an antioxidant compound to reduce a ferric oxidant (Fe3+) to a

14 ferrous complex (Fe2+) by electron-transfer, and this indicated the capacity of the compound to

15 reduce reactive species [21]. The findings indicated that the CFR fraction possessed the highest

16 value (49.17 mg TE.g-1), followed by CF70 (22.86 mg TE.g-1) and then CF50 (16.08 mg TE.g-1).

17 In comparison to antioxidant activities of other compounds (fucoxanthin and phenolics) from

18 H.banksii reported in our previous study [25], these polysaccharides exhibited lower antioxidant

19 activities. Low free radical scavenging activities of sulfated polysaccharides were also found in

20 some algae (Dictyota mertensis (8.7%), Dictyota menstruallis (7.5%), Gracilaria caudata (8.0%)

21 and Caulerpa sertularioides (11.8%)) compared to gallic acid of 93.7% [29]. From the findings,

22 it is noted that phenolics as residues were mainly responsible for antioxidant activity of the 1 polysaccharide fractions from H.banksii; and the sulfate content and molecular weight of the

2 polysaccharides fractions also significantly affected this activity.

3 3.3 Cytotoxic effect of the fractions against pancreatic cancer

4 Marine algae derived polysaccharides have been shown to possess the growth inhibitory activity

5 against several cancer cell lines [30, 31] and the molecular mechanisms for anticancer effects of

6 fucoidans have been outlined [32]. In our study, the polysaccharide fractions from brown alga H.

7 banksii were investigated for the activity against three pancreatic cell lines (MiaPaCa2, BxPC3,

8 CFPAC1) and non-tumorigenic cells (HPDE) using the CCK-8 assay at the serial concentrations

9 (25- ȝJP/-1). The findings showed that all polysaccharide fractions dose-dependently

10 inhibited pancreatic cancer cell lines in vitro and the results are all presented in Table 3.

11 In regard to MiaPaCa2 cells, the CFR fraction exhibited high cytotoxic activity (inhibition >

12 60%) at concentrations •  ȝJP/-1. There was no significant difference in the inhibitory

13 efficacies of the CFR fractions at the concentrations of 100-150 ȝJP/-1 (P>0.05). The CF50

14 and CF70 fractions possessed lower inhibitory activities compared to the CFR fraction. The

15 inhibition efficacy of the fractions was characterised by IC50 values with 140.79; 130.66 and

16 ȝJP/-1 for the CF50; CF70 and CFR fractions, respectively (Table 4). For the BxPC3 cell

17 line, the suppressions of cancer cell growth by the CF70 and CFR fractions were found to be

18 efficacious (inhibition ! DWWKHFRQFHQWUDWLRQV•ȝJP/-1. The inhibitory activity of the

19 CFR fraction was observed to be higher than that of the CF50 and CF70 fractions at all

20 concentrations. However, these fractions showed lower cytotoxicity against cancer cells

21 compared to standard gemcitabine (50 nM) with the inhibition of 75.03%. With the secondary

22 pancreatic cancer cell line (CFPAC1), the inhibitory efficacies of both CF70 and CFR fractions

23 ZHUHRYHUDWWKHGRVDJHV•ȝJP/-1 and the trend was similar to MiaCaPa2 and BxPC3 1 as activity of the CFR fraction was higher than those of the CF50 and CF70 fractions. The lower

-1 2 IC50 value of the CFR fraction (65.71 ȝJP/ ) against the CFPAC1 cells compared to the CF50

3 ȝJP/-1) and &) ȝJP/-1) fractions. From the findings, it can be seen that the

4 polysaccharide fractions possessed high inhibitory activity • against MiaPaCa2, BxPC3

5 and CFPAC1 cells at the FRQFHQWUDWLRQV •  ȝJP/-1); however, were significantly lower

6 inhibition compared to gemcitabine (50nM).

7 In the HPDE cells, it was shown that the CF50 and CF70 fractions were less toxic to the normal

-1 8 cells (IC50 = DQGȝJP/ , respectively; Table 4). Importantly, the CF70 fraction

9 was more selective cytotoxicity than the CF50 fraction (higher activity of the CF70 fraction

10 against cancer cells but less toxic to the normal cells compared to those of the CF50 fraction). On

-1 11 the other hand, the stronger toxicity was found in the CFR fraction with the IC50 = 45.50 ȝJP/

12 compared to those of the CF50 and CF70 fractions. The CFR fraction was highly toxic towards

13 the QRUPDOFHOOV LQKLELWLRQRI HYHQDWDORZFRQFHQWUDWLRQRIȝJP/-1. It is thought

14 that the high toxicity of the CFR fraction was due to its high proportion of phenolics (Table 1).

15 The results were supported by Athukorala et al. [13] who indicated that high proportion of

16 phenolics in the phenolic (CphF) fraction from the Ecklonia cava extract resulted in higher anti-

17 proliferative activity against cancer cells compared to the polysaccharide (CpoF) fraction.

18 The structural properties of polysaccharides (sulfate content, monosaccharide content, molecular

19 weight…) significantly affected their biological activities. From the findings (Table 3), the cell

20 growth inhibitory activity of the CF70 fraction was significantly higher than that of the CF50

21 fraction at almost all concentrations. The reason for this may be that the CF70 fraction possessed

22 lower molecular polysaccharides than those of the CF50 fraction (high molecular

23 polysaccharides were precipitated by low ethanol percentage, while precipitation of lower 1 molecular polysaccharides required higher ethanol ratio in the solution). It was in line with Yang

2 et al. [33] who pointed out that lower molecular weight fucoidans from Undaria pinnatifida

3 enhanced the cancer cell inhibition activity by hydrolysing fucoidans with mild conditions

4 (microwave irradiation, acid solution) compared to original fucoidans. Cytotoxic activity of low

5 weight fucoidans from Fucus vesiculosus obtained by gamma-irradiation against cancers was

6 improved compared to that of native fucoidans [7]. On the other hand, the sulfate content of

7 polysaccharides also affected their anticancer activity. It could be that the cell growth inhibition

8 of the CF70 fraction was higher compared to that of the CF50 fraction due to higher sulfate

9 content. It was agreement with Costa et al. [29] that the sulfate content had a positive correlation

10 with anti-proliferative efficacies of the polysaccharide fractions from tropical algae. The increase

11 in sulfate groups of fucoidans enhanced their antiangiogenic activities [34] and improved anti-

12 proliferative activities against fibroblast cell line (CCL39) [35]. In addition, it was demonstrated

13 that the role of sulfate groups in fucoidans related to anticancer activity was higher compared to

14 monosaccharide content in the algal extracts [36]. From the findings, it is proposed that cytotoxic

15 activity of the polysaccharides from H.banksii against pancreatic cancer affected by their sulfate

16 content and molecular weight.

17 Sulfated polysaccharides from marine algae against various cancer cell lines were summarised in

18 the previous studies [30, 32]. Fucoidans from Turbinaria conoides indicated high activity against

19 human lung cancer cells (A549), while low toxicity to normal Vero cells (the CTC50 values; 45

20 and 325 μg.mL-1, respectively) [37]. The human colon cancer cells (HCT-15) were significantly

-1 21 suppressed by fucoidan (IC50 = 75 μg.mL )from Sargassum cinereum [38]. Until now, there

22 was little information about the activity of marine polysaccharides against pancreatic cancer

23 reported. Sulfated polysaccharides from Turbinaria conoides showed significantly inhibitory 1 effects on the pancreatic cancer cell lines (MiaPaCa-2 and Panc-1) in both dose-dependent and

2 time-dependent manner; and inhibited angiogenesis [39]. In comparison to other compounds

3 (fucoxanthin and phenolics) from the H.banksii extract from our study, sulfated polysaccharides

4 showed lower inhibitory activities against pancreatic cancer cell lines (data not shown).

5 However, our findings and the previous studies have demonstrated potential applications of

6 marine polysaccharides due to their selective cytotoxicity against various cancer cells [37, 40,

7 41].

8 From the results, it could be suggested that the polysaccharides from the H.bankssi extract

9 should be further purified and purified polysaccharides will be assessed for the cytotoxic effects,

10 molecular mechanisms against pancreatic cancer cell lines. The combination of polysaccharides

11 and other compounds (fucoxanthin, phenolics or standard of gemcitabine) with different ratios

12 should be estimated to enhance anticancer efficacy and reduce side-effects to the normal cells.

13 Furthermore, chemotherapy and radiotherapy are considered as the standard treatment

14 approaches for patients with unresectable, metastatic or locally advanced inoperable pancreatic

15 cancer. These findings have demonstrated the potential of the sulfated polysaccharides (the CF50

16 and CF70 fractions) from brown alga H. banksii against pancreatic cancer.

17 Conclusions

18 Three polysaccharide fractions were isolated from the H.banksii extract with the significant

19 differences in yield, chemical profile (TPC, total sugar, sulfate content). The antioxidant

20 activities (ABTS, DPPH and FRAP) were observed to be low. These fractions exhibited high

21 cytotoxic effects (cancer cell growth inhibition of 39.35–82.82% at concentrations of 100–200

22 μg.ml-1) against pancreatic cancer cell lines. The CF50 and CF70 fractions showed a low toxicity

23 towards the normal cells, while high toxicity was found in the CFR fraction. Therefore, 1 polysaccharides in the CF50 and CF70 fractions will be purified and evaluated for anticancer

2 efficacies, apoptotic and cell cycle mechanisms; and further their potential applications in the

3 functional food and pharmaceutical industries.

4

5

6 Acknowledgements

7 The authors kindly thank to University of Newcastle; the Vietnamese Government through the

8 Ministry of Education and Training; the Ministry of Agriculture and Rural Development

9 awarding a VIED-TUIT scholarship to Thanh Trung DANG.

10

11

12

13

14 Conflict of interest

15 The authors declare no conflict of interest.

16

17

18

19

20 1 References

2 1. El Gamal AA. Biological importance of marine algae. Saudi Pharm J 2010; 18: 1-25.

3 2. Gupta S, Abu-Ghannam N. Bioactive potential and possible health effects of edible brown

4 seaweeds. Trend Food Sci Technol 2011; 22: 315-326.

5 3. Stengel DB, Connan S, Popper ZA. Algal chemodiversity and bioactivity: sources of natural

6 variability and implications for commercial application. Biotechnol Adv 2011; 29: 483-501.

7 4. Ale MT, Meyer AS. Fucoidans from brown seaweeds: An update on structures, extraction

8 techniques and use of enzymes as tools for structural elucidation. RSC Adv 2013; 3: 8131-

9 8141.

10 5. Jo BW, Choi SK. Degradation of fucoidans from Sargassum fulvellum and their biological

11 activities. Carbohyd Polym 2014; 111: 822-829.

12 6. Matsubara K, Xue C, Zhao X, Mori M, Sugawara T, Hirata T. Effects of middle molecular

13 weight fucoidans on in vitro and ex vivo angiogenesis of endothelial cells. Inter J Mol Med

14 2005; 15: 695-699.

15 7. Choi JI, Kim HJ. Preparation of low molecular weight fucoidan by gamma-irradiation and

16 its anticancer activity. Carbohyd Polym 2013; 97: 358-362.

17 8. Yang C, Chung D, You S. Determination of physicochemical properties of sulphated fucans

18 from sporophyll of Undaria pinnatifida using light scattering technique. Food Chem 2008;

19 111: 503-507.

20 9. Behrens D, Walther W, Fichtner I. Pancreatic cancer models for translational research.

21 Pharmacol Therapeut 2017; 173: 146-158. 1 10. Verbeke C, Löhr M, Karlsson JS, Del Chiaro M. Pathology reporting of pancreatic cancer

2 following neoadjuvant therapy: challenges and uncertainties. Cancer Treat Rev 2015; 41:

3 17-26.

4 11. Xu Q, Zhang TP, Zhao YP. Advances in early diagnosis and therapy of pancreatic cancer.

5 Hepatob Pancreat Dis Inter 2011; 10: 128-135.

6 12. Scarlett CJ, Salisbury EL, Biankin AV, Kench J. Precursor lesions in pancreatic cancer:

7 morphological and molecular pathology. Pathol 2011; 43: 183-200.

8 13. Athukorala Y, Kim KN, Jeon YJ. Antiproliferative and antioxidant properties of an

9 enzymatic hydrolysate from brown alga, Ecklonia cava. Food Chem Toxicol 2006; 44:

10 1065-1074.

11 14. Dang TT, Vuong QV, Schreider MJ, Bowyer MC, Van Altena IA, Scarlett CJ. Optimisation

12 of ultrasound-assisted extraction conditions for phenolic content and antioxidant activities of

13 the alga Hormosira banksii using response surface methodology. J Appl Phycol 2017; 29:

14 3161-3173.

15 15. Dodgson K, Price R. A note on the determination of the ester sulphate content of sulphated

16 polysaccharides. Biochem J 1962; 84: 106.

17 16. Mak W, Hamid N, Liu T, Lu J, White W. Fucoidan from New Zealand Undaria pinnatifida:

18 Monthly variations and determination of antioxidant activities. Carbohyd Polym 2013; 95:

19 606-614.

20 17. Dubois M, Gilles KA, Hamilton JK, Rebers PT, Smith F. Colorimetric method for

21 determination of sugars and related substances. Anal Chem 1956; 28: 350-356. 1 18. Peasura N, Laohakunjit N, Kerdchoechuen O, Vongsawasdi P, Chao LK. Assessment of

2 biochemical and immunomodulatory activity of sulphated polysaccharides from Ulva

3 intestinalis. Inter J Biol Macromol 2016; 91: 269-277.

4 19. Thaipong K, Boonprakob U, Crosby K, Cisneros-Zevallos L, Hawkins Byrne D.

5 Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity

6 from guava fruit extracts. J Food Compo Anal 2006; 19: 669-675.

7 20. Brand-Williams W, Cuvelier M, Berset C. Use of a free radical method to evaluate

8 antioxidant activity. LWT-Food Sci Technol 1995; 28: 25-30.

9 21. Benzie I, Strain J. Ferric reducing/antioxidant power assay: direct measure of total

10 antioxidant activity of biological fluids and modified version for simultaneous measurement

11 of total antioxidant power and ascorbic acid concentration. Method Enzymol 1999; 15-27.

12 22. Saravana PS, Tilahun A, Gerenew C, Tri VD, Kim NH, Kim GD, Chun BS. Subcritical

13 water extraction of fucoidan from Saccharina japonica: optimization, characterization and

14 biological studies. J Appl Phycol 2017; 1-12.

15 23. Liu X, Liu B, Wei XL, Sun ZL, Wang CY. Extraction, Fractionation, and Chemical

16 Characterisation of Fucoidans from the Brown Seaweed Sargassum pallidum. Czech J Food

17 Sci 2016; 34:

18 24. Saravana PS, Cho YJ, Park YB, Woo HC, Chun BS. Structural, antioxidant, and emulsifying

19 activities of fucoidan from Saccharina japonica using pressurized liquid extraction.

20 Carbohyd Polym 2016; 153: 518-525.

21 25. Dang TT, Bowyer MC, Van Altena IA, Scarlett CJ. Comparison of chemical profile and

22 antioxidant properties of the brown algae. Inter J Food Sci Technol 2018; 53: 174-181. 1 26. Lim SJ, Aida WMW, Maskat MY, Mamot S, Ropien J, Mohd DM. Isolation and antioxidant

2 capacity of fucoidan from selected Malaysian seaweeds. Food Hydrocolloid 2014; 42: 280-

3 288.

4 27. Wang CY, Wu TC, Hsieh SL, Tsai YH, Yeh CW, Huang CY. Antioxidant activity and

5 growth inhibition of human colon cancer cells by crude and purified fucoidan preparations

6 extracted from Sargassum cristaefolium. J Food Drug Anal 2015; 23: 766-777.

7 28. Zhang Q, Yu P, Li Z, Zhang H, Xu Z, Li P. Antioxidant activities of sulfated polysaccharide

8 fractions from Porphyra haitanesis. J Appl Phycol 2003; 15: 305-310.

9 29. Costa L, Fidelis G, Cordeiro S, Oliveira R, Sabry D, Câmara R, Farias, E. Biological

10 activities of sulfated polysaccharides from tropical seaweeds. Biomed Pharm 2010; 64: 21-

11 28.

12 30. Fedorov SN, Ermakova SP, Zvyagintseva TN, Stonik VA. Anticancer and cancer preventive

13 properties of marine polysaccharides: some results and prospects. Mar Drugs 2013; 11:

14 4876-4901.

15 31. Senthilkumar K, Manivasagan P, Venkatesan J, Kim SK. Brown seaweed fucoidan:

16 Biological activity and apoptosis, growth signaling mechanism in cancer. Inter J Biol

17 Macromol 2013; 60: 366-374.

18 32. Wu L, Sun J, Su X, Yu Q, Yu Q, Zhang P. A review about the development of fucoidan in

19 antitumor activity: Progress and challenges. Carbohyd Polym 2016; 154: 96-111.

20 33. Yang C, Chung D, Shin IS, Lee H, Kim J, Lee Y, You S. Effects of molecular weight and

21 hydrolysis conditions on anticancer activity of fucoidans from sporophyll of Undaria

22 pinnatifida. Inter J Biol Macromol 2008; 43: 433-437. 1 34. Koyanagi S, Tanigawa N, Nakagawa H, Soeda S, Shimeno H. Oversulfation of fucoidan

2 enhances its anti-angiogenic and antitumor activities. Biochem Pharm 2003; 65: 173-179.

3 35. Haroun-Bouhedja F, Ellouali M, Sinquin C, Boisson-Vidal C. Relationship between sulfate

4 groups and biological activities of fucans. Thrombosis Res 2000; 100: 453-459.

5 36. Ale MT, Maruyama H, Tamauchi H, Mikkelsen JD, Meyer AS. Fucoidan from Sargassum

6 sp. and Fucus vesiculosus reduces cell viability of lung carcinoma and melanoma cells in

7 vitro and activates natural killer cells in mice in vivo. Inter J Biol Macromol 2011; 49: 331-

8 336.

9 37. Marudhupandi T, Kumar TTA, Lakshmanasenthil S, Suja G, Vinothkumar T. In vitro

10 anticancer activity of fucoidan from Turbinaria conoides against A549 cell lines. Inter J Biol

11 Macromol 2015; 72: 919-923.

12 38. Somasundaram SN, Shanmugam S, Subramanian B, Jaganathan R. Cytotoxic effect of

13 fucoidan extracted from Sargassum cinereum on colon cancer cell line HCT-15. Inter J Biol

14 Macromol 2016; 91: 1215-1223.

15 39. Delma CR, Somasundaram ST, Srinivasan GP, Khursheed M, Bashyam MD, Aravindan N.

16 Fucoidan from Turbinaria conoides: A multifaceted ‘deliverable’to combat pancreatic

17 cancer progression. Inter J Biol Macromol 2015; 74: 447-457.

18 40. Alwarsamy M, Gooneratne R, Ravichandran R. Effect of fucoidan from Turbinaria conoides

19 on human lung adenocarcinoma epithelial (A549) cells. Carbohyd Polym 2016; 152: 207-

20 213.

21 41. Xue M, Ge Y, Zhang J, Wang Q, Hou L, Liu Y, Li Q. Anticancer properties and

22 mechanisms of fucoidan on mouse breast cancer in vitro and in vivo. PloS one 2012; 7:

23 e43483. Fucoidan Total sulfate Total sugar Total phenolic content Yield (%) fractions content (%) content (%) (mg GAE.g-1) CF50 30.53 10.59±0.16a 29.31±1.64c 25.59±0.65a CF70 5.82 12.19±0.05b 23.21±1.08b 26.79±0.11a CFR 9.56 11.00±1.05ab 7.37±0.15a 90.77±0.68b Table 1: Chemical profile of the polysaccharide fractions from H.banksii. All values are means ± standard deviation (n=3) and those in the same column not sharing the same superscript letter are significantly different from the others (P<0.05). Fucoidan Antioxidant activities of fractions (mgTE.g-1) fractions ABTS DDPH FRAP CF50 12.40±0.16a 8.66±0.62a 16.08±0.49a CF70 17.49±0.13b 15.29±0.78b 22.86±0.18b CFR 106.99±0.69c 103.05±0.84c 49.17±0.44c Table 2: Antioxidant activities of the fucoidan fractions from H.banksii. All values are means ± standard deviation (n=3) and those in the same column not sharing the same superscript letter are significantly different from the others (P<0.05). Concentrations of the fractions (μg.mL-1) and gemcitabine (50 nM) Cell Lines Fractions 200 150 100 50 25 Gem. CF50 35.2±3.46d 35.61±4.99d 27.26±6.70b 20.78±4.30b 8.47±3.21a HDPE CF70 35.03±2.86e 29.32±3.25d 20.07±4.67b 15.06±6.21b 7.52±2.19a 89.67±0.98c CFR 91.63±3.78c 90.00±1.31c 89.65±4.40c 60.66±3.50b 25.4±3.23a CF50 57.04±2.24d 56.49±2.72d 39.35±2.65c 21.31±2.14b 11.93±3.11a MiaCaPa2 CF70 63.61±2.65e 49.47±2.33d 42.64±1.39c 28.31±2.91b 13.35±5.23a 87.28±1.59f CFR 78.33±3.19d 64.47±3.14c 62.85±2.04c 46.41±1.57b 29.11±3.76a CF50 57.97±5.13d 45.72±6.03c 43.82±4.82c 28.49±8.98b 13.35±2.92a BxPC3 CF70 59.04±1.71d 53.97±2.05c 51.34±5.83c 29.22±7.78b 9.58±5.54a 75.03±1.28e CFR 69.8±3.33d 68.64±3.92d 53.57±2.21c 32.12±3.43b 10.95±6.38a CF50 72.35±0.88e 62.96±1.93d 40.82±2.74c 27.17±3.15b 8.56±6.29a CFPAC1 CF70 73.97±2.99d 65.61±2.59c 63.38±3.72c 36.47±4.04b 20.12±4.67a 92.42±2.34f CFR 82.82±1.67d 70.19±3.24c 68.61±2.43c 41.25±1.93b 20.35±5.21a Table 3: The cytotoxic activities of the fucoidan fractions at different concentrations compared to gemcitabine (50nM) in human pancreatic ductal epithelial (HDPE) and pancreatic cancer cell lines. All values are means ± standard deviation (n=3) and those in the same row not sharing the same superscript letter are significantly different from the others (P<0.05). IC values (μg.mL-1) Cell lines 50 CF50 CF70 CFR HPDE 526.32 781.25 42.50 MiaCapa-2 140.79 130.66 61.89 BxPC-3 91.32 121.12 90.78 CFPAC-1 105.76 76.46 65.71 -1 Table 4: IC50 values (μg.mL ) of the polysaccharide fractions from H.banksii on pancreatic cancer cell lines. PART 3: GENERAL DISCUSSION AND CONCLUSIONS

3.1 General discussion

This thesis is divided into three main parts. Part 1 and 2 outline preparative methods for optimal drying and extraction of brown algae; and the isolation of key bioactive constituents

(fucoxanthin, phlorotannins and polysaccharides), respectively. Part 3 evaluates the antioxidant properties and cytotoxic activities of the isolated algal compounds against a range of pancreatic cancer cell lines. In a study of sample preparation, it is necessary to investigate the impact of different drying conditions (sun, microwave, oven, vacuum, de- humidification and freeze drying) on the final product quality (phytochemical profile and antioxidant activities). For the extraction and optimisation study, the parameters (solvent type and ratio, temperature, time, irradiation power) associated with two extraction processes - ultrasonic water bath (UAE) and a microwave oven (MAE) were examined for phenolic yield and antioxidant activities. With the separation of compounds, the use of separation and isolation techniques (thin layer chromatography, column chromatography, partition, centrifugation and HPLC) were utilised to achieve high yield and product purity.

Finally, the individual and groups of compounds were evaluated for biological activities

(antioxidant and cytotoxic activities) and potent applications of bioactive compounds from marine algae for food, pharmaceutical and other industries.

Relevant information on marine algae has been discussed in the introduction section, and the main findings of these studies have been highlighted in the synopsis of the eight research papers (section 2.1). Therefore, general discussions and recommendations are outlined in the following sections.

39

3.1.1 Preparation of the samples (drying and extraction)

It is undoubted that an effective drying process is vital for the preparation of natural product samples, such that the physico-chemical profile and antioxidants are preserved for subsequent extraction and isolation. It is noted that each drying method has inherent advantages and drawbacks (Bennamoun et al., 2015), with choice of method being subject to a range of parameters, including temperature, time, drying properties, energy costs and the structure and quantity of the material undergoing treatment. A traditional way of drying materials is sun drying, which is highly cost effective because of low capital costs (Brink &

Marx, 2013). The process however takes a long time to complete and is subject to weather constraints which can lead to degradation of target compounds through photochemical and oxidative processes (Chan et al., 1997). Freeze drying usually yields better quality dried product as a consequence of the low temperatures used that eliminates losses associated with compound degradation (Wong & Cheung, 2001).

Paper II represents the first study to comprehensively evaluate six drying methods (sun, microwave, oven, vacuum, de-humidification and freeze drying) for the brown alga H. banksii. The findings show that vacuum and dehumidification drying at a temperature of 50

°C and freeze drying are chosen for drying algae with high quality final products related to yields, moisture resident, chemical profile (TPC, TFC and tannins) and antioxidant activities

(ABTS, DPPH and FRAP). The findings were consistent with previous work by Djaeni and

Sari (2015) and Hossain et al., (2010) who similarly found that de-humidification and vacuum drying at temperature (70 °C) and relative humidity (~ 10%) were effective drying conditions. Microwave assisted drying of algae, while efficient (in terms of time) led to a dramatic reduction in the yield of extracted biological compounds, presumably as a consequence of thermal degradation by microwave irradiation (Lim & Murtijaya, 2007) and

40 was thus deemed an unsuitable drying process. The findings were in agreement with previous studies that freeze drying is the best for drying plants and algae. However, the use of freeze drying is considered too expensive for the large scale.

The properties of organic solvents are important for successful and optimal extraction of bioactives from marine and plants species. A range of organic solvents have been historically used to extract bioactive compounds from plant materials because of their excellent solvating properties. Many are, however, toxic in nature and highly flammable, posing significant health and safety concerns. Minimisation of hazardous reagents is required for all processes to restrict the harmful effects not only for humans but also for environment (Hashemi et al., 2018). Ethanol as a solvent for extraction of natural active compounds is safer than acetone, methanol or other organic solvents. Although flammable, it has relatively low toxicity and is suitable for use in most food and pharmaceutical processes (Ritchie, 2006).

In this study, aqueous ethanol (70%) was chosen as the solvent for the extraction of phenolics from a brown alga H.banksii by UAE (Paper III) and from S.vestitum using

MAE (Paper IV). Pure ethanol was used for the extraction of fucoxanthin from the

H.banksii extract (Paper VI). The study found aqueous ethanol to be a good absorber of microwave radiation and an effective and safe solvent for the extraction of phytochemicals

(Zhou & Liu, 2006). Sulfated polysaccharides (fucoidans) were extracted by water and isolated by pure ethanol (Paper VIII). It is clear that in comparison to organic solvents, water is considered to be a better option due to its non-toxic, non-corrosive and non- flammable. Water is also a cheap, abundant and environmentally friendly solvent for the extraction of high polarity compounds such as phenolics and polysaccharides.

41

(Polshettiwar & Varma, 2008). However, most organic substrates are insoluble in water, and evaporation of water for drying samples is not an energy-efficient process.

The effectiveness of two extraction methods - ultrasound assisted extraction and microwave assisted extraction were evaluated in terms of the phenolic yield and antioxidant activities.

Ultrasound assisted extraction and microwave assisted extraction for bioactive compounds are cost effective extraction techniques compared to processes such as pressurised liquid

H[WUDFWLRQ 3/(  VXSHUFULWLFDO ÀXLG H[WUDFWLRQ 6)(  HQ]\PH DVVLVWHG H[WUDFtion (EAE) with high costs of solvents, enzymes and equipment (Zhang et al., 2011). For ultrasonic extraction, a number of important factors such as power of ultrasound, extraction temperature and time, solvents, the ratios of solvents to solid and time affected chemical profile and antioxidant activities of the plant extracts (Chemat & Khan, 2011; Vilkhu et al.,

2008). In comparison to microwave assisted extraction, using an ultrasonic water bath is not restricted by type of solvents, moisture content of materials and controlling temperature that suitable for extraction of thermo-labile compounds (Chemat & Khan, 2011).

Ultrasound assisted extraction (UAE) has been demonstrated to enhance extraction efficacy of bioactive compounds, through low solvent use and energy consumption, as well as reduced processing time (Roselló-Soto et al., 2015; Shirsath et al., 2012). Paper III describes optimal conditions for UAE for phenolics and antioxidant activities. For the extraction of alga H.banksii, the mathematical model created using RSM was shown to be a reliable predictor of the influence of independent variables (temperature, time and power) phenolic yield and extract antioxidant activity. The findings were in agreement with previous studies which reported that low temperature and low power was suitable for UAE of thermo-sensitive compounds in the extract (Le Lan et al., 2008; Teh and Birch et al.,

2014). H.banksii extracts showed a positive relationship between TPC and antioxidant

42 assays and was consistent with findings by Amorim et al. (2012); Le Lan et al. (2008) and

Matanjun et al. (2008). The findings in this study pointed out that higher yield of phenolics and higher antioxidant activities of the H.banksii extract could be obtained with the optimal parameters, including low temperature of 30 °C, time of 60 min and power of 60% compared those of conventional methods. The findings supported the hypothesis, which is presented in section 1.5.

It has been shown that microwave assisted extraction has some important merits such asshorter extraction time, higher extraction yield and less solvent consumption, and is an energy saving technique compared to conventional extraction methods. However, some disadvantages of microwave assisted extraction are that extraction efficacy is affected by the ability of solvents to absorb microwaves. The target compounds and solvents are required to be non-polar and it is not fit for extraction of thermally labile compounds (Chan et al., 2011;

Zhang et al., 2011).

It was the similar trend to UAE of H.banksii that the RSM mathematical models were fit for predicting effects of independent variances (irradiation times, ethanol percentage and microwave power) on the yield and antioxidant activity of the S.vestitum extract using microwave assisted extraction (Paper IV). Yield of phenolics and antioxidant activities of the S.vestitum extract were the most affected by the percentage of ethanol. The phenolic recovery yield and antioxidant activities of extraction using pure ethanol as a solvent were lower than those of 70% ethanol. It is likely due to swelling of materials caused by water that made the bonds of the material’s structure weak and/or broken and compounds were easily released from the material (Guo et al., 2001; Xiao et al., 2008). Maximum TPC was obtained from the alga Saccharina japonica using a 55% aqueous ethanol extract (He et al.,

2013) while a percentage range of 10-30% was applied in the case of Monostroma nitidum

43

(Lin et al., 2013) extraction. Both irradiation time and power were found to affect the activity of thermo-sensitive compounds in the S. vestitum extracts, suggesting that higher power and shorter extraction times produced optimal outcomes. The optimal microwave assisted extraction conditions for S.vestitum achieved were ethanol concentration of 70%, irradiation time of 75 seconds and power of 80% or 840 watts (Paper IV). Therefore, the use of ultrasound and microwave irradiation techniques was suitable for extraction of high yields of phenolics from brown algae with high antioxidant activities.

3.1.2 Bioactive compounds and isolation processes from brown algae

Generally, marine algae are considered as a rich source of bioactive compounds with purported health benefits and potential application in the functional food and pharmaceutical industries. It is crucial to evaluate the chemical profile and biological activities of algal extracts for further isolation of valuable compounds. Therefore, it is desirable to compare the chemical profiles and antioxidant activities of the six algal species, which is shown in

Paper I, for the selection of key species for further processing and investigation.

There was a positive correlation between total phenolic compounds and antioxidant activities of algal extracts (Matanjun et al., 2008). Brown algae have been demonstrated to have antioxidant capacity comparatively higher compared to red and green algae in the group of marco-algae (Balboa et al., 2013). According to the results of Paper I, the highest

TPC and TFC values was observed in H.banksii, while Padina sp. possessed the highest levels of tannins. TPC values from S.vestitum and Padina sp. were significantly higher compared to the remaining algal species. In addition, phenolics from these extracts were shown to be mainly responsible for antioxidant activities. Therefore, three out of six algal species investigated (S.vestitum, H.banksii and Padina sp.) with potential phenolics were selected for further processing.

44

Fucoxanthin is one of the most abundant pigments of brown algae with amounts estimated to be around 10% of total carotenoids in nature (Rajauria et al., 2016). The structure of this pigment found in brown algae is almost all trans-fucoxanthin (Jaswir et al., 2013; Nakazawa et al., 2009). From the findings described in Paper I, fucoxanthin content was observed in all six brown algae and quantitated by a UV-visible spectrophotometer. This pigment also possessed high antioxidant activities. However, due to lower amounts of fucoxanthin, the role of fucoxanthin was not vital compared the phenolics in the antioxidant activities from the algal extracts tested (Paper I). Fucoxanthin was separated by preparative thin layer chromatography or/and column chromatography with high purity. The mobile phases used for isolation process were mixtures of organic solvents with types of solvents and their proportions in the mixtures depended on algal species (Jaswir et al., 2011; Noviendri et al.,

2011; Rajauria et al., 2016).

Fucoxanthin from H.banksii was extracted by pure ethanol and successfully isolated via column chromatography with amount of 0.58 mg Fx.g-1 and purity of 92.3% by HPLC analysis. While being isolated in only small quantities, fucoxathin derived from H. banksii, exhibited high inhibitory activity against pancreatic cancer cell lines (Paper VI). This finding was consistent with previous studies which (Kumar et al., 2013; Rengarajan et al.,

2013; Tanaka et al., 2012) found activity against several other cancer cell lines.

As discussed above, phlorotannins play a crucial role in antioxidant activities of brown algae. The partition technique is often applied to separating phlorotannins with different polarities in the organic fractions. The higher purity phlorotannins were obtained as they are continuously isolated through column chromatography in the sub-fractions (Cho et al.,

2011; Li et al., 2009). From the TLC analysis presented in Paper V, it may be that the structures of phlorotannins were similar, resulting in difficulties in separation of individual

45 compounds (the bands of phlorotannins were close or overlapped on the TLC plates). From

TLC analysis, and chemical and antioxidant properties of fractions, it was suggested that the partition technique was efficient in the isolation of phenolics with different polarities. The ethyl acetate fraction was reported to possess high amount of phenolics and antioxidant activities from several brown algae (Chakraborty et al., 2013; Duan et al., 2006; Wang et al.,

2012). The crude extract of H.banksii was partitioned to give five fractions (Paper V). The ethyl acetate fraction possessed higher TPC and tannins, while higher TFC in the hexane fraction compared to the crude extract and other fractions. The antioxidant activities of the ethyl acetate fraction were also found to be the highest compared to those of other fractions.

Polysaccharides (fucoidans) successfully isolated from marine algae were reported in several earlier studies (De Souza et al., 2007; Ermakova et al., 2013; Palanisamy et al.,

2017). Water is popularly used to extract polysaccharides from marine algae. However, to make the polysaccharides with greater purity, the algal powders should be treated with pure ethanol, methanol or acetone to remove the pigments, lipids, proteins and other low molecular weight compounds (Ye et al., 2008). These algal biomasses were then extracted with water to obtain the polysaccharides. From the extracts, polysaccharides were separated using pure ethanol to precipitate them with different concentrations of ethanol in the extracts. It was reported that at the low concentration of ethanol in the extracts (about 20%), alginic acid was precipitated and the range of ethanol concentrations (20 – 65%) was suitable to isolate fucoidan, while ethanol percentage over 65% was used to separate laminarin (Anno et al., 1966). From the alga H.banksii, three polysaccharide fractions

(CF50, CF70 and CFR) were separated (Paper VIII). It was shown that sulfated polysaccharides (fucoidans) were observed in all fractions. The highest sulfate ratio was

12.19% in the CF70 fraction and these values for the CF50 and CFR fraction were 10.59

46 and 11.00%, respectively. The CF70 fraction had higher antioxidant activities compared to others. Three fractions showed low antioxidant activities compared to those of fucoxanthin and phenolics from the same alga (H.banksii) reported in our earlier studies (Papers I and

V). The sulfate ratios of the fractions were reported to significantly affect biological activities of the polysaccharide fractions. The polysaccharide fractions from Porphyra haitanesis with high sulfate content exhibited higher antioxidant activities compared to others (Zhang et al., 2003). The high sulfate content was shown to have a positive correlation with anti-proliferative efficacies in the polysaccharide fractions against cancer cells (Costa et al., 2010).

3.1.3 Activity of algal compounds against pancreatic cancer cell lines

Marine algae are a rich source of fucoxanthin and possess potential human health benefits.

There is also increasing attention of fucoxanthin due to its reported supporting role in the treatment of chronic diseases, such as diabetes and cancers (D’Orazio et al., 2012; Tanaka et al., 2012). Results from Paper VI indicated that purified fucoxanthin from H.banksii possessed high cytotoxic activities against several pancreatic cancer cell lines (MiaPaCa-2,

BxPC-3 and CFPAC-1) at concentrations of 100 – 200 μg.mL-1, while showing medium cytotoxicity against non-tumourigenic cells (HPDE) in vitro. Fucoxanthin in the gastrointestinal tract is converted to fucoxanthiol by digestive enzymes and then amarouciaxanthin A in the liver. The mechanisms of fucoxanthiol and/or amarouciaxanthin

A (forms of fucoxathin in animals) are suggested to be the regulatory effect of fucoxanthin on biomolecules related to apoptosis and the cell cycle (D’Orazio et al., 2012). Interestingly, fucoxanthin showed medium cytotoxicity against normal cells in vitro tests but it was found to be safe for in vivo tests with fucoxanthin treated mice (Beppu et al., 2009; Geisen et al.,

2015). Until now, little information about the cytotoxic effects of fucoxanthin against

47 pancreatic cancer cells has been reported (nitro-fucoxanthins, reaction products of fucoxanthin and peroxinitrite, with high inhibition against pancreatic cancers were presented in Paper VI). From the findings, fucoxanthin from H.banksii may be a promising compound for further the investigations into novel treatment agents for pancreatic cancers.

The findings from Paper VII demonstrated that the phenolic-enriched fractions (butanol and ethyl acetate) of the H.banksii showed high cytotoxicity against pancreatic cancer cell lines. However, the polar phenolics (the butanol fraction) were found show low toxicity to normal cells compared to the medium polar phenolics (the ethyl acetate fraction). In this paper, it was shown that almost all crude extracts and phenolic-containing fractions derived from brown algae possessed cytotoxic activity (76 – 100%) against selected pancreatic cancer cell lines (McCauley et al. 2015; Aravidan et al., 2013). Results from the Hormosira banksii extract also suggest that phenolic-enriched fractions play a significant role in both antioxidant and cytotoxic activity against pancreatic cancer. Currently, there is only limited information relating to the activity of the crude extracts, fractions, individual phenolic compounds, pigments, polysaccharides and other components from brown algae against pancreatic cancer. However, several active compounds from other marine sources with high activity against pancreatic cancers were reported in several previous studies. Batzellines and pyrroloiminoquinones alkaloids derived from the Caribbean sponge Batzella sp exhibit selective cytotoxicity towards the pancreatic cancer cell lines AsPC-1, Panc-1, BxPC-3, and

MIA PaCa2 (IC50 values < 10 μM) and low cytotoxicity to the normal cells. These compounds showed higher inhibition compared to 5-fluorouracil, the current benchmark treatment for pancreatic cancer (Guzmán et al., 2009). Butenolides, D IDPLO\ RI Įȕ- unsaturated lactones, from a marine-derived fungus Aspergillus terreus with high cytotoxic activities (IC50; 5.3–9.4 μM) against pancreatic ductal adenocarcinoma cells (PANC-1) were

48 observed via apoptotic body formation, membrane blebbing, cell shrinkage and nuclear condensation of cancer cells (Qi et al., 2018). Manzamine A, a member of the manzamine alkaloids, from marine sponges of the genus Haliclona is a potential inhibitor of autophagy

(with low concentration of 10 μM) that is essential for pancreatic tumor growth by preventing autophagosome turnover (Kallifatidis et al., 2013). Leiodermatolide, a polyketide macrolide, from a sponge of the genus Leiodermatium, indicated selective cytotoxicity toward the AsPC-1, BxPC-3 and MIA PaCa-2 cells. Induction of apoptosis by leiodermatolide was observed in these cell lines. This compound also induces cell cycle arrest without effect on in vitro polymerization or depolymerization of tubulin alone and enhances polymerization of tubulin containing microtubule associated proteins (Guzmán et al., 2016). Other marine sources (kahalalide F from the mollusc Elysia rubefescens; squalamine, from the dogfish shark Squalusacanthias; neovastat, a derivative of shark cartilage extract) have been developed to the stage of clinical trials against several cancers

(Nobili et al., 2009).

It was shown that three polysaccharide fractions (CF50, CF70 and CFR) from the alga

H.banksii showed high cytotoxic activities against pancreatic cancer cell lines (Paper VIII).

The inhibitory activity of the CFR fraction was significantly higher than these of the CF50 and CF70 fractions. It could be that the CF70 fraction was found to be higher in antioxidant and cytotoxic activities than these of the CF50 due to higher sulfate content. However the

CFR fraction was toxic to the normal cells due to the high amount of phenolic residue.

Therefore, the CF50 and CF70 fractions showed potential for the development of novel treatment agents for pancreatic cancer. The polysaccharide fraction from the Ecklonia cava extract showed activity against cancer cells, but was lower compared to that of the phenolic fraction (Athukorala et al., 2006). Yang et al. (2008a) revealed that lower molecular weight fucoidans from Undaria pinnatifida enhanced cancer cell inhibitory activity by hydrolysing 49 native fucoidans with mild conditions (microwave irradiation, acid solution). The sulfate polysaccharides from the Turbinaria conoides extract showed significant inhibitory effects on both the pancreatic cancer cell lines, MiaPaCa-2 and Panc-1 cells in a dose-dependent and time-dependent manner (Delma et al., 2015). From the results (Paper VIII), it could be suggested that the polysaccharide fractions from the H.banksii extract should be further purified and assessed for their cytotoxic effects against pancreatic cancer cell lines. In addition, these polysaccharides were safe and showed low toxicity to the normal cells; however, phenolics exhibited excellent inhibition of cancer cell growth with high toxicity towards non-tumorigenic cells.

3.1 Conclusions and recommendations

3.1.1 Conclusions

In the thesis, samples of six brown algae (Sargassum vestitum; Sargassum linearifolium;

Phyllospora comosa; Padina sp.; Hormosira banksii; Sargassum podocanthum) were prepared (drying, extraction and isolation of components) for assessment of their biological activity as antioxidant properties and cytotoxic activity against a range of pancreatic cancer cell lines.

Preliminary assessment of six algal species found three (Sargassum vestitum; Padina sp. and Hormosira banksii) possessed high constituent yields of bioactives and high antioxidant activity and were consequently selected for detailed study (Paper I). H. banksii was assessed in greatest detail and was found to possess three principle compounds of interest

(fucoxanthin, phenolics and polysaccharides). S. vestitum and Padina sp. were found to be rich sources of phenolics but were not investigated further because of time constraints.

Three drying methods of vacuum, de-humidification and freeze drying were suitable for the preparation of algal samples (H.banksii, Paper II). Temperature of 40 – 50 °C was found to 50 be optimal for the drying of H.banksii. The active compounds (fucoxanthin, phenolics) were found to be sensitive to higher temperatures and oxygen levels.

The algal extracts were obtained using ultrasound assisted extraction (H.banksii) and microwave assisted extraction (S.vestitum). The optimal conditions determined via response surface methodology to generate high yields of phenolics and antioxidant activities of the

H.banksii extracts were presented in Paper III (temperature of 30 °C, ultrasound power of

60% and a extraction time of 60 min.), and for S.vestitum in Paper IV (high ethanol concentration - 70%, short irradiation time - 75 s and medium microwave power - 80%).

The findings from Paper V showed that isolation of phenolics from the H.banksii was most efficient using solvent-partitioning extraction. The solvent ratios for ethyl acetate and butanol fractions in the crude extract were 11.61 and 7.78% (w/w), respectively. These were the phlorotannin-enriched fractions that showed higher antioxidant activity compared to other fractions, and the crude extract, even higher than that of standard compounds (BHT, ascorbic acid and tocopherol) at some concentrations.

The cytotoxic activities of algal compounds from H.banksii against pancreatic cancer cell lines (MiaPaCa-2, BxPC-3 and CFPAC-1) were presented in three papers (Paper VI, VII and VIII). In Paper VI, fucoxanthin content was successfully isolated via column chromatography and quantitated by HPLC analysis with fucoxanthin content of 0.58 - 0.61 mg Fx.g-1 alga (w/w) checked by UV-visible spectrophotometer and HPLC analysis. The high cell growth inhibition by fucoxanthin against pancreatic cancer cells was observed at concentrations of 100 – 200 μg.mL-1. The cytotoxic activities against pancreatic cancer cells by phlorotannins, as presented in Paper VII, demonstrated that phenolics in the ethyl

DFHWDWHIUDFWLRQVLJQLILFDQWO\LQKLELWHGQXPHURXVFDQFHUFHOOVW\SHV LQKLELWLRQHIILFDFLHV•

80% with all cell lines). For pancreatic cancer, both the ethyl acetate and butanol fractions

51 exhibited high inhibitory effects against cancer cells. Interestingly, the phenolics in the butanol fraction possessed selective cytotoxicity against pancreatic cancer cell lines.

Polysaccharides (fucoidans) from H.banksii were extracted by water using an ultrasonic water bath, as presented in Paper VIII. Three polysaccharide fractions (the CF50, CF70 and

CFR fractions) exhibited high cytotoxic effects on pancreatic cancer cell lines, and both the

CF50 and CF70 fractions demonstrated low toxicity towards non-tumorigenic cells.

3.1.2 Recommendations

Based on the results obtained from this study, the author recommends the following studies on brown algae:

x The polar phenolic compounds of the butanol fraction from alga H.banksii should be

isolated and assessed for activities against pancreatic cancer cell lines.

x Fucoidans from fucoidan enriched fractions from alga H.banksii should be purified to

enhance their observed activities against pancreatic cancer cell lines.

x Phenolic compounds from alga S.vestitum should be assessed for activities against

pancreatic cancer cell lines.

x The combinations between chemotherapeutic agents (such as gemcitabine), in

combination with fucoxanthin, phenolic compounds and fucoidan should be assessed

for activities against pancreatic cancer cell lines.

x Fucoxanthin, phenolic compounds and fucoidan should be assessed for their

mechanisms of action (apoptosis/cell cycle) against pancreatic cancer cell lines.

52

BIBLIOGRAPHY

Ahn, G.N., Kim, K.N., Cha, S.H., Song, C.B., Lee, J., Heo, M.S., Kim, J.S. (2007).

Antioxidant activities of phlorotannins purified from Ecklonia cava on free radical

scavenging using ESR and H2O2-mediated DNA damage. European Food Research

and Technology, 226(1-2), 71-79.

Ale, M. T., Maruyama, H., Tamauchi, H., Mikkelsen, J. D., & Meyer, A. S. (2011).

Fucoidan from Sargassum sp. and Fucus vesiculosus reduces cell viability of lung

carcinoma and melanoma cells in vitro and activates natural killer cells in mice in vivo.

International Journal of Biological Macromolecules, 49(3), 331-336.

Alekseyenko, T., Zhanayeva, S. Y., Venediktova, A., Zvyagintseva, T., Kuznetsova, T.,

Besednova, N., & Korolenko, T. (2007). Antitumor and antimetastatic activity of

fucoidan, a sulfated polysaccharide isolated from the Okhotsk Sea Fucus evanescens

brown alga. Bulletin of Experimental Biology and Medicine, 143(6), 730-732.

Amsler, C. D., & Fairhead, V. A. (2005). Defensive and sensory chemical ecology of brown

algae. Advances in Botanical Research, 43, 1-91.

Anno, K., Terahata, H., Hayashi, Y., & Seno, N. (1966). Isolation and purification of

fucoidin from brown seaweed Pelvetia wrightii. Agricultural and Biological Chemistry,

30(5), 495-499.

Aravindan, S., Delma, C. R., Thirugnanasambandan, S. S., Herman, T. S., & Aravindan, N.

(2013). Anti-pancreatic cancer deliverables from sea: first-hand evidence on the

efficacy, molecular targets and mode of action for multifarious polyphenols from five

different brown-algae. PloS one, 8(4), e61977.

Aryee, A. N., Agyei, D., & Akanbi, T. O. (2018). Recovery and utilization of seaweed

pigments in food processing. Current Opinion in Food Science 19 113-119.

53

Athukorala, Y., Kim, K.N., & Jeon, Y.J. (2006). Antiproliferative and antioxidant properties

of an enzymatic hydrolysate from brown alga, Ecklonia cava. Food and Chemical

Toxicology, 44(7), 1065-1074.

Azmir, J., Zaidul, I., Rahman, M., Sharif, K., Mohamed, A., Sahena, F., Omar, A. (2013).

Techniques for extraction of bioactive compounds from plant materials: a review.

Journal of Food Engineering, 117(4), 426-436.

Balboa, E. M., Conde, E., Moure, A., Falqué, E., & Domínguez, H. (2013). In vitro

antioxidant properties of crude extracts and compounds from brown algae. Food

Chemistry, 138(2), 1764-1785.

Barros Gomes Camara, R., Silva Costa, L., Pereira Fidelis, G., Duarte Barreto Nobre, L. T.,

Dantas-Santos, N., Lima Cordeiro, S., Oliveira Rocha, H. A. (2011). Heterofucans from

the brown seaweed Canistrocarpus cervicornis with anticoagulant and antioxidant

activities. Marine Drugs, 9(1), 124-138.

%Dú' %R\DFÕ,+  0RGHOLQJDQGRSWLPL]DWLRQ,8VDELOLW\RIUHVSRQVHVXUIDFH

methodology. Journal of Food Engineering, 78(3), 836-845.

Batista, A. P., Raymundo, A., Sousa, I., & Empis, J. (2006). Rheological characterization of

coloured oil-in-water food emulsions with lutein and phycocyanin added to the oil and

aqueous phases. Food Hydrocolloids, 20(1), 44-52.

Bayraktar, S., Bayraktar, U. D., & Rocha-Lima, C. M. (2010). Recent developments in

palliative chemotherapy for locally advanced and metastatic pancreas cancer. World

Journal of Gastroenterology: WJG, 16(6), 673.

Bennamoun, L., Afzal, M. T., & Léonard, A. (2015). Drying of alga as a source of

bioenergy feedstock and food supplement–A review. Renewable and Sustainable

Energy Reviews, 50, 1203-1212.

54

Beppu, F., Niwano, Y., Tsukui, T., Hosokawa, M., & Miyashita, K. (2009). Single and

repeated oral dose toxicity study of fucoxanthin (FX), a marine carotenoid, in mice. The

Journal of Toxicological Sciences, 34(5), 501-510.

Berthod, A., Ruiz-Angel, M., & Carda-Broch, S. (2009). Countercurrent chromatography:

People and applications. Journal of Chromatography A, 1216(19), 4206-4217.

Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, L. A. (2008).

Response surface methodology (RSM) as a tool for optimization in analytical

chemistry. Talanta, 76(5), 965-977.

Bocanegra, A., Bastida, S., Benedi, J., Rodenas, S., & Sánchez-Muniz, F. J. (2009).

Characteristics and nutritional and cardiovascular-health properties of seaweeds.

Journal of Medicinal Food, 12(2), 236-258.

Brink, J., & Marx, S. (2013). Harvesting of Hartbeespoort Dam micro-algal biomass

through sand filtration and solar drying. Fuel, 106, 67-71.

Brown, E. M., Allsopp, P. J., Magee, P. J., Gill, C. I., Nitecki, S., Strain, C. R., & McSorley,

E. M. (2014). Seaweed and human health. Nutrition Reviews, 72(3), 205-216.

Bucar, F., Wube, A., & Schmid, M. (2013). Natural product isolation–how to get from

biological material to pure compounds. Natural Product Reports, 30(4), 525-545.

Burris, H. r., Moore, M. J., Andersen, J., Green, M. R., Rothenberg, M. L., Modiano, M. R.,

Tarassoff, P. (1997). Improvements in survival and clinical benefit with gemcitabine as

first-line therapy for patients with advanced pancreas cancer: a randomized trial.

Journal of Clinical Oncology, 15(6), 2403-2413.

Cabrita, M. T., Vale, C., & Rauter, A. P. (2010). Halogenated compounds from marine

algae. Marine Drugs, 8(8), 2301-2317.

Cai, W., Gu, X., & Tang, J. (2008). Extraction, purification, and characterization of the

polysaccharides from Opuntia milpa alta. Carbohydrate Polymers, 71(3), 403-410. 55

Cai, Z., Lee, F., Wang, X., & Yu, W. (2002). A capsule review of recent studies on the

application of mass spectrometry in the analysis of Chinese medicinal herbs. Journal of

Mass Spectrometry, 37(10), 1013-1024.

Chakraborty, K., Praveen, N. K., Vijayan, K. K., & Rao, G. S. (2013). Evaluation of

phenolic contents and antioxidant activities of brown seaweeds belonging to Turbinaria

spp.(Phaeophyta, Sargassaceae) collected from Gulf of Mannar. Asian Pacific Journal

of Tropical Biomedicine, 3(1), 8-16.

Chan, C.H., Yusoff, R., Ngoh, G.C., & Kung, F.W.L. (2011). Microwave-assisted

extractions of active ingredients from plants. Journal of Chromatography A, 1218(37),

6213-6225.

Chan, J. C.C., Cheung, P. C.K., & Ang, P. O. (1997). Comparative studies on the effect of

three drying methods on the nutritional composition of seaweed Sargassum

hemiphyllum (Turn.) C. Ag. Journal of Agricultural and Food Chemistry, 45(8), 3056-

3059.

Chandini, S. K., Ganesan, P., & Bhaskar, N. (2008). In vitro antioxidant activities of three

selected brown seaweeds of India. Food Chemistry, 107(2), 707-713.

Chemat, F., & Khan, M. K. (2011). Applications of ultrasound in food technology:

processing, preservation and extraction. Ultrasonics Sonochemistry, 18(4), 813-835.

Cho, M., Kang, I.J., Won, M.-H., Lee, H.S., & You, S. (2010). The antioxidant properties of

ethanol extracts and their solvent-partitioned fractions from various green seaweeds.

Journal of Medicinal Food, 13(5), 1232-1239.

Cho, M., Lee, H.S., Kang, I.J., Won, M.H., & You, S. (2011). Antioxidant properties of

extract and fractions from Enteromorpha prolifera, a type of green seaweed. Food

Chemistry, 127(3), 999-1006.

56

Claessens, H., & Van Straten, M. (2004). Review on the chemical and thermal stability of

stationary phases for reversed-phase liquid chromatography. Journal of

Chromatography A, 1060(1-2), 23-41.

Coleman, M. A., Kelaher, B. P., Steinberg, P. D., & Millar, A. J. (2008). Absence of a large

brown macroalga on urbanized rocky reefs around Sydney, Australia, and evidence for

historical decline 1. Journal of Phycology, 44(4), 897-901.

Costa, L., Fidelis, G., Cordeiro, S., Oliveira, R., Sabry, D., Câmara, R., Farias, E. (2010).

Biological activities of sulfated polysaccharides from tropical seaweeds. Biomedicine &

Pharmacotherapy, 64(1), 21-28.

Craft, B. D., Kerrihard, A. L., Amarowicz, R., & Pegg, R. B. (2012). Phenol-based

antioxidants and the in vitro methods used for their assessment. Comprehensive

Reviews in Food Science and Food Safety, 11(2), 148-173.

Cragg, G. M., & Pezzuto, J. M. (2016). Natural products as a vital source for the discovery

of cancer chemotherapeutic and chemopreventive agents. Medical Principles and

Practice, 25(Suppl. 2), 41-59.

D’Orazio, N., Gemello, E., Gammone, M. A., de Girolamo, M., Ficoneri, C., & Riccioni, G.

(2012). Fucoxantin: A treasure from the sea. Marine Drugs, 10(3), 604-616.

Dang, T. T., Bowyer, M. C., Van Altena, I. A., & Scarlett, C. J. (2017). Comparison of

chemical profile and antioxidant properties of the brown algae. International Journal of

Food Science & Technology.

De Souza, M. C. R., Marques, C. T., Dore, C. M. G., da Silva, F. R. F., Rocha, H. A. O., &

Leite, E. L. (2007). Antioxidant activities of sulfated polysaccharides from brown and

red seaweeds. Journal of Applied Phycology, 19(2), 153-160.

57

Dellai, A., Laajili, S., Le Morvan, V., Robert, J., & Bouraoui, A. (2013). Antiproliferative

activity and phenolics of the Mediterranean seaweed Laurencia obusta. Industrial

Crops and Products, 47, 252-255.

Delma, C. R., Somasundaram, S. T., Srinivasan, G. P., Khursheed, M., Bashyam, M. D., &

Aravindan, N. (2015). Fucoidan from Turbinaria conoides: A multifaceted

‘deliverable’to combat pancreatic cancer progression. International Journal of

Biological Macromolecules, 74, 447-457.

Djaeni, M., & Sari, D. A. (2015). Low Temperature Seaweed Drying Using Dehumidified

Air. Procedia Environmental Sciences, 23, 2-10.

Dorsey, J. G., & Dill, K. A. (1989). The molecular mechanism of retention in reversed-

phase liquid chromatography. Chemical Reviews, 89(2), 331-346.

Duan, X.J., Zhang, W.W., Li, X.M., & Wang, B.G. (2006). Evaluation of antioxidant

property of extract and fractions obtained from a red alga, Polysiphonia urceolata.

Food Chemistry, 95(1), 37-43.

Eom, S.H., Kim, Y.-M., & Kim, S.K. (2012). Antimicrobial effect of phlorotannins from

marine brown algae. Food and Chemical Toxicology, 50(9), 3251-3255.

Ermakova, S., Men'shova, R., Vishchuk, O., Kim, S.-M., Um, B.-H., Isakov, V., &

Zvyagintseva, T. (2013). Water-soluble polysaccharides from the brown alga Eisenia

bicyclis: Structural characteristics and antitumor activity. Algal Research, 2(1), 51-58.

Fairhead, V. A., Amsler, C. D., McClintock, J. B., & Baker, B. J. (2005). Variation in

phlorotannin content within two species of brown macroalgae (Desmarestia anceps and

D. menziesii) from the Western Antarctic Peninsula. Polar Biology, 28(9), 680-686.

Fedorov, S. N., Ermakova, S. P., Zvyagintseva, T. N., & Stonik, V. A. (2013). Anticancer

and cancer preventive properties of marine polysaccharides: some results and prospects.

Marine Drugs, 11(12), 4876-4901. 58

Foley, S. A., Szegezdi, E., Mulloy, B., Samali, A., & Tuohy, M. G. (2011). An

unfractionated fucoidan from Ascophyllum nodosum: extraction, characterization, and

apoptotic effects in vitro. Journal of Natural Products, 74(9), 1851-1861.

Foo, S. C., Yusoff, F. M., Ismail, M., Basri, M., Chan, K. W., Khong, N. M., & Yau, S. K.

(2015). Production of fucoxanthin-rich fraction (FxRF) from a diatom, Chaetoceros

calcitrans (Paulsen) Takano 1968. Algal Research, 12, 26-32.

Fung, A., Hamid, N., & Lu, J. (2013). Fucoxanthin content and antioxidant properties of

Undaria pinnatifida. Food Chemistry, 136(2), 1055-1062.

Ganesan, P., Matsubara, K., Sugawara, T., & Hirata, T. (2013). Marine algal carotenoids

inhibit angiogenesis by down-regulating FGF-2-mediated intracellular signals in

vascular endothelial cells. Molecular and Cellular Biochemistry, 380(1-2), 1-9.

Garson, M. (1989). Biosynthetic studies on marine natural products. Nat. Prod. Rep., 6(2),

143-170.

Geisen, U., Zenthoefer, M., Peipp, M., Kerber, J., Plenge, J., Managò, A., Adam, D. (2015).

Molecular mechanisms by which a Fucus vesiculosus extract mediates cell cycle

inhibition and cell death in pancreatic cancer cells. Marine Drugs, 13(7), 4470-4491.

Gellerstedt, G. (1992). Gel permeation chromatography Methods in Lignin Chemistry (pp.

487-497): Springer.

Guo, Z., Jin, Q., Fan, G., Duan, Y., Qin, C., & Wen, M. (2001). Microwave-assisted

extraction of effective constituents from a Chinese herbal medicine Radix puerariae.

Analytica Chimica Acta, 436(1), 41-47.

Gupta, S., & Abu-Ghannam, N. (2011). Bioactive potential and possible health effects of

edible brown seaweeds. Trends in Food Science & Technology, 22(6), 315-326.

Güven, K. C., Percot, A., & Sezik, E. (2010). Alkaloids in marine algae. Marine Drugs,

8(2), 269-284. 59

Guzmán, E. A., Johnson, J. D., Carrier, M. K., Meyer, C. I., Pitts, T. P., Gunasekera, S. P.,

& Wright, A. E. (2009). Selective cytotoxic activity of the marine derived batzelline

compounds against pancreatic cancer cell lines. Anti-cancer Drugs, 20(2), 149.

Guzmán, E. A., Xu, Q., Pitts, T. P., Mitsuhashi, K. O., Baker, C., Linley, P. A., Suh, E. M.

(2016). Leiodermatolide, a novel marine natural product, has potent cytotoxic and

antimitotic activity against cancer cells, appears to affect microtubule dynamics, and

exhibits antitumor activity. International Journal of Cancer, 139(9), 2116-2126.

Hashemi, B., Zohrabi, P., & Dehdashtian, S. (2018). Application of green solvents as

sorbent modifiers in sorptive-based extraction techniques for extraction of

environmental pollutants. TrAC Trends in Analytical Chemistry, 119 50-61.

He, Z., Chen, Y., Chen, Y., Liu, H., Yuan, G., Fan, Y., & Chen, K. (2013). Optimization of

the microwave-assisted extraction of phlorotannins from Saccharina japonica Aresch

and evaluation of the inhibitory effects of phlorotannin-containing extracts on HepG2

cancer cells. Chinese Journal of Oceanology and Limnology, 31, 1045-1054.

Hobbs, A. N., & Young, R. J. (2013). Practical purification of hydrophilic fragments and

lead/drug-like molecules by reverse phase flash chromatography: tips, tricks and

contemporary developments. Drug Discovery Today, 18(3-4), 148-154.

Holdt, S. L., & Kraan, S. (2011). Bioactive compounds in seaweed: functional food

applications and legislation. Journal of Applied Phycology, 23(3), 543-597.

Hosokawa, M., Kudo, M., Maeda, H., Kohno, H., Tanaka, T., & Miyashita, K. (2004).

)XFR[DQWKLQLQGXFHVDSRSWRVLVDQGHQKDQFHVWKHDQWLSUROLIHUDWLYHHIIHFWRIWKH33$5Ȗ

ligand, troglitazone, on colon cancer cells. Biochimica et Biophysica Acta (BBA)-

General Subjects, 1675(1), 113-119.

60

Hosokawa, M., Wanezaki, S., Miyauchi, K., Kurihara, H., Kohno, H., Kawabata, J.,

Takahashi, K. (1999). Apoptosis-Inducing Effect of Fucoxanthin on Human Leukemia

Cell Line HL-60. Food Science and Technology Research, 5(3), 243-246.

Hossain, M., Barry-Ryan, C., Martin-Diana, A. B., & Brunton, N. (2010). Effect of drying

method on the antioxidant capacity of six Lamiaceae herbs. Food Chemistry, 123(1),

85-91.

Hossain, Z., Kurihara, H., Hosokawa, M., & Takahashi, K. (2005). Growth inhibition and

induction of differentiation and apoptosis mediated by sodium butyrate in Caco-2 cells

with algal glycolipids. In Vitro Cellular & Developmental Biology-Animal, 41(5-6),

154-159.

Hou, Y., Wang, J., Jin, W., Zhang, H., & Zhang, Q. (2012). Degradation of Laminaria

japonica fucoidan by hydrogen peroxide and antioxidant activities of the degradation

products of different molecular weights. Carbohydrate Polymers, 87(1), 153-159.

Huang, X.Y., Ignatova, S., Hewitson, P., & Di, D.-L. (2016). An overview of recent

progress in elution mode of counter current chromatography. TrAC Trends in Analytical

Chemistry, 77, 214-225.

Hung, S. W., Mody, H. R., & Govindarajan, R. (2012). Overcoming nucleoside analog

chemoresistance of pancreatic cancer: a therapeutic challenge. Cancer letters, 320(2),

138-149.

Imbs, T. I., Ermakova, S. P., Fedoreyev, S. A., Anastyuk, S. D., & Zvyagintseva, T. N.

(2013). Isolation of fucoxanthin and highly unsaturated monogalactosyldiacylglycerol

from brown alga Fucus evanescens C Agardh and in vitro investigation of their

antitumor activity. Marine Biotechnology, 15(5), 606-612.

61

Isbrucker, R. A., Guzman, E. A., Pitts, T. P., & Wright, A. E. (2009). Early effects of

lasonolide A on pancreatic cancer cells. Journal of Pharmacology and Experimental

Therapeutics, 331(2), 733-739.

Ito, Y. (2005). Golden rules and pitfalls in selecting optimum conditions for high-speed

counter-current chromatography. Journal of Chromatography A, 1065(2), 145-168.

Itoh, H., Noda, H., Amano, H., Zhuaug, C., Mizuno, T., & Ito, H. (1993). Antitumor activity

and immunological properties of marine algal polysaccharides, especially fucoidan,

prepared from Sargassum thunbergii of Phaeophyceae. Anticancer Research, 13(6A),

2045-2052.

Iwai, K. (2008). Antidiabetic and antioxidant effects of polyphenols in brown alga Ecklonia

stolonifera in genetically diabetic KK-Ay mice. Plant Foods for Human Nutrition,

63(4), 163.

Jandera, P. (2011). Stationary and mobile phases in hydrophilic interaction chromatography:

a review. Analytica Chimica Acta, 692(1-2), 1-25.

Jaswir, I., Noviendri, D., Salleh, H. M., Taher, M., & Miyashita, K. (2011). Isolation of

fucoxanthin and fatty acids analysis of Padina australis and cytotoxic effect of

fucoxanthin on human lung cancer (H1299) cell lines. African Journal of

Biotechnology, 10(81), 18855-18862.

Jaswir, I., Noviendri, D., Salleh, H. M., & Miyashita, K. (2012). Fucoxanthin extractions of

brown seaweeds and analysis of their lipid fraction in methanol. Food Science and

Technology Research, 18(2), 251-257.

Jaswir, I., Noviendri, D., Salleh, H. M., Taher, M., Miyashita, K., & Ramli, N. (2013).

Analysis of fucoxanthin content and purification of all-trans-fucoxanthin from

Turbinaria turbinata and Sargassum plagyophyllum by SiO2 open column

62

chromatography and reversed phase-HPLC. Journal of Liquid Chromatography &

Related Technologies, 36(10), 1340-1354.

Jeon, S. M., Kim, H. J., Woo, M. N., Lee, M. K., Shin, Y. C., Park, Y. B., & Choi, M. S.

(2010). Fucoxanthin-rich seaweed extract suppresses body weight gain and improves

lipid metabolism in high-fat-fed C57BL/6J mice. Biotechnology Journal, 5(9), 961-969.

Joana Gil-Chávez, G., Villa, J. A., Fernando Ayala-Zavala, J., Basilio Heredia, J.,

Sepulveda, D., Yahia, E. M., & González-Aguilar, G. A. (2013). Technologies for

extraction and production of bioactive compounds to be used as nutraceuticals and food

ingredients: an overview. Comprehensive Reviews in Food Science and Food Safety,

12(1), 5-23.

Jungbauer, A., & Hahn, R. (2009). Ion-exchange chromatography Methods in Enzymology

(Vol. 463, pp. 349-371): Elsevier.

Kadam, S., & Prabhasankar, P. (2010). Marine foods as functional ingredients in bakery and

pasta products. Food Research International, 43(8), 1975-1980.

Kadam, S. U., Tiwari, B. K., & O’Donnell, C. P. (2013). Application of Novel Extraction

Technologies for Bioactives from Marine Algae. Journal of Agricultural and Food

Chemistry, 61(20), 4667-4675.

Kadam, S. U., Tiwari, B. K., Smyth, T. J., & O’Donnell, C. P. (2015). Optimization of

ultrasound assisted extraction of bioactive components from brown seaweed

Ascophyllum nodosum using response surface methodology. Ultrasonics

Sonochemistry, 23, 308-316.

Kala, H. K., Mehta, R., Sen, K. K., Tandey, R., & Mandal, V. (2016). Critical analysis of

research trends and issues in microwave assisted extraction of phenolics: Have we

really done enough. TrAC Trends in Analytical Chemistry, 85, 140-152.

63

Kallifatidis, G., Hoepfner, D., Jaeg, T., Guzmán, E. A., & Wright, A. E. (2013). The marine

natural product manzamine A targets vacuolar ATPases and inhibits autophagy in

pancreatic cancer cells. Marine Drugs, 11(9), 3500-3516.

Kasai, A., Arafuka, S., Koshiba, N., Takahashi, D., & Toshima, K. (2015). Systematic

synthesis of low-molecular weight fucoidan derivatives and their effect on cancer cells.

Organic & Biomolecular Chemistry, 13(42), 10556-10568.

Kendrick, G. A., Harvey, E., Wernberg, T., Harman, N., & Goldberg, N. (2004). The role of

disturbance in maintaining diversity of benthic macroalgal assemblages in southwestern

Australia. Japanese Journal of Phycology, 52, 5-9.

Kevekordes, K., & Clayton, M. N. (2000). Development of Hormosira banksii

(Phaeophyceae) embryos in selected components of secondarily-treated sewage

effluent. Journal of Phycology, 36(1), 25-32.

Khanavi, M., Nabavi, M., Sadati, N., Shams Ardekani, M., Sohrabipour, J., Nabavi, S. M.

B., Ostad, S. N. (2010). Cytotoxic activity of some marine brown algae against cancer

cell lines. Biological Research, 43(1), 31-37.

Kim, E. J., Park, S. Y., Lee, J.Y., & Park, J. H. Y. (2010). Fucoidan present in brown algae

induces apoptosis of human colon cancer cells. BMC Gastroenterology, 10(1), 96.

Kim, K.N., Ahn, G., Heo, S.J., Kang, S.M., Kang, M.C., Yang, H.M., Jeon, B.T. (2013).

Inhibition of tumor growth in vitro and in vivo by fucoxanthin against melanoma

B16F10 cells. Environmental Toxicology and Pharmacology, 35(1), 39-46.

Kim, K.N., Heo, S.J., Kang, S.M., Ahn, G., & Jeon, Y.J. (2010). Fucoxanthin induces

apoptosis in human leukemia HL-60 cells through a ROS-mediated Bcl-xL pathway.

Toxicology in Vitro, 24(6), 1648-1654.

64

Kim, M.M., Van Ta, Q., Mendis, E., Rajapakse, N., Jung, W.-K., Byun, H.G., Kim, S.K.

(2006). Phlorotannins in Ecklonia cava extract inhibit matrix metalloproteinase activity.

Life sciences, 79(15), 1436-1443.

Kim, S. M., Shang, Y. F., & Um, B. H. (2011). A preparative method for isolation of

fucoxanthin from Eisenia bicyclis by centrifugal partition chromatography.

Phytochemical Analysis, 22(4), 322-329.

.RQtFþNRYi 5 9DQĖNRYi . 9DQtNRYi - 9iQĖRYi . 0XFKRYi / 6XEKDQRYi ,

.ROiUĜ0  $QWL-cancer effects of blue-green alga Spirulina platensis, a natural

source of bilirubin-like tetrapyrrolic compounds. Annals of Hepatology: Official

Journal of the Mexican Association of Hepatology, 13(2).

Konishi, I., Hosokawa, M., Sashima, T., Kobayashi, H., & Miyashita, K. (2006).

Halocynthiaxanthin and fucoxanthinol isolated from Halocynthia roretzi induce

apoptosis in human leukemia, breast and colon cancer cells. Comparative Biochemistry

and Physiology Part C: Toxicology & Pharmacology, 142(1-2), 53-59.

Kotake-Nara, E., Asai, A., & Nagao, A. (2005). Neoxanthin and fucoxanthin induce

apoptosis in PC-3 human prostate cancer cells. Cancer Letters, 220(1), 75-84.

Kotake-Nara, E., Terasaki, M., & Nagao, A. (2005). Characterization of apoptosis induced

by fucoxanthin in human promyelocytic leukemia cells. Bioscience, Biotechnology, and

Biochemistry, 69(1), 224-227.

Koyanagi, S., Tanigawa, N., Nakagawa, H., Soeda, S., & Shimeno, H. (2003). Oversulfation

of fucoidan enhances its anti-angiogenic and antitumor activities. Biochemical

Pharmacology, 65(2), 173-179.

Kumar, S. R., Hosokawa, M., & Miyashita, K. (2013). Fucoxanthin: A Marine Carotenoid

Exerting Anti-Cancer Effects by Affecting Multiple Mechanisms. Marine Drugs,

11(12), 5130-5147. 65

Kusaykin, M., Bakunina, I., Sova, V., Ermakova, S., Kuznetsova, T., Besednova, N.,

Zvyagintseva, T. (2008). Structure, biological activity, and enzymatic transformation of

fucoidans from the brown seaweeds. Biotechnology Journal, 3(7), 904-915.

Lee, S.-H., & Jeon, Y.J. (2013). Anti-diabetic effects of brown algae derived phlorotannins,

marine polyphenols through diverse mechanisms. Fitoterapia, 86, 129-136.

Lee, S.H., Qian, Z.J., & Kim, S.-K. (2010). A novel angiotensin I converting enzyme

inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in

spontaneously hypertensive rats. Food Chemistry, 118(1), 96-102.

Li, C., Wang, C., Wang, S., Qian, G., Zhu, Q., Liu, Y., & Wang, W. (2013). Optimization of

ultrasonic-assisted extraction technology of Sargassum fusiforme polysaccharides and

evaluation of their antioxidant activity. Food Science and Technology Research, 19(2),

157-162.

Li, J., Wientjes, M. G., & Au, J. L.S. (2010). Pancreatic cancer: pathobiology, treatment

options, and drug delivery. The AAPS Journal, 12(2), 223-232.

Li, Y.-X., Himaya, S., & Kim, S.K. (2013). Triterpenoids of marine origin as anti-cancer

agents. Molecules, 18(7), 7886-7909.

Li, Y.-X., Wijesekara, I., Li, Y., & Kim, S.-K. (2011). Phlorotannins as bioactive agents

from brown algae. Process Biochemistry, 46(12), 2219-2224.

Li, Y., Qian, Z.J., Ryu, B., Lee, S.-H., Kim, M.-M., & Kim, S.K. (2009). Chemical

components and its antioxidant properties in vitro: An edible marine brown alga,

Ecklonia cava. Bioorganic & Medicinal Chemistry, 17(5), 1963-1973.

Liazid, A., Palma, M., Brigui, J., & Barroso, C. G. (2007). Investigation on phenolic

compounds stability during microwave-assisted extraction. Journal of Chromatography

A, 1140(1), 29-34.

66

Lim, S., Cheung, P., Ooi, V., & Ang, P. (2002). Evaluation of antioxidative activity of

extracts from a brown seaweed, Sargassum siliquastrum. Journal of Agricultural and

Food Chemistry, 50(13), 3862-3866.

Lim, Y., & Murtijaya, J. (2007). Antioxidant properties of Phyllanthus amarus extracts as

affected by different drying methods. LWT-Food Science and Technology, 40(9), 1664-

1669.

Lin, Y.P., Huang, S.L., & Wu, S.C. (2013). Effects of Microwave-assisted Extraction on the

Free Radical Scavenging and Ferrous Chelating Abilities of Porphyra dentate Extract.

Marine Science and Technology, 38(4), 317-328.

Lin, Y.P., Wu, S.C., & Huang, S.-L. (2013). Effects of Microwave-Assisted Extraction on

the Free Radical Scavenging and Ferrous Chelating Abilities of Monostroma Nitidum

Extract. Journal of Marine Science and Technology, 21(5), 611-617.

Liu, M., Hansen, P. E., & Lin, X. (2011). Bromophenols in marine algae and their

bioactivities. Marine Drugs, 9(7), 1273-1292.

Liu, X., Liu, B., Wei, X.L., Sun, Z.L., & Wang, C.Y. (2016). Extraction, Fractionation, and

Chemical Characterisation of Fucoidans from the Brown Seaweed Sargassum pallidum.

Czech Journal of Food Science, 34(5) 406 - 413.

Lordan, S., Ross, R. P., & Stanton, C. (2011). Marine bioactives as functional food

ingredients: potential to reduce the incidence of chronic diseases. Marine Drugs, 9(6),

1056-1100.

Macías-Sánchez, M., Mantell, C., Rodriguez, M., de la, Martínez de la Ossa, E., Lubián, L.,

& Montero, O. (2009). Comparison of supercritical fluid and ultrasound-assisted

extraction of carotenoids and chlorophyll a from Dunaliella salina. Talanta, 77(3), 948-

952.

67

Maeda, H., Hosokawa, M., Sashima, T., & Miyashita, K. (2007). Dietary combination of

fucoxanthin and fish oil attenuates the weight gain of white adipose tissue and

decreases blood glucose in obese/diabetic KK-Ay mice. Journal of Agricultural and

Food Chemistry, 55(19), 7701-7706.

Mak, W., Hamid, N., Liu, T., Lu, J., & White, W. (2013). Fucoidan from New Zealand

Undaria pinnatifida: Monthly variations and determination of antioxidant activities.

Carbohydrate Polymers, 95(1), 606-614.

Mantyka, C. S., & Bellwood, D. R. (2007). Macroalgal grazing selectivity among

herbivorous coral reef fishes. Marine Ecology Progress Series, 352, 177-185.

Marston, A., & Hostettmann, K. (1991). Modern separation methods. Natural Product

Reports, 8(4), 391-413.

Martin, L. J. (2015). Fucoxanthin and its metabolite fucoxanthinol in cancer prevention and

treatment. Marine Drugs, 13(8), 4784-4798.

Marzinelli, E., Campbell, A. H., Vergés, A., Coleman, M., Kelaher, B. P., & Steinberg, P.

(2014). Restoring seaweeds: does the declining fucoid Phyllospora comosa support

different biodiversity than other habitats? Journal of Applied Phycology, 26(2), 1089-

1096.

Matanjun, P., Mohamed, S., Mustapha, N. M., Muhammad, K., & Ming, C. H. (2008).

Antioxidant activities and phenolics content of eight species of seaweeds from north

Borneo. Journal of Applied Phycology, 20(4), 367-373.

Matsubara, K., Xue, C., Zhao, X., Mori, M., Sugawara, T., & Hirata, T. (2005). Effects of

middle molecular weight fucoidans on in vitro and ex vivo angiogenesis of endothelial

cells. International Journal of Molecular Medicine, 15(4), 695-699.

68

May, D., & Clayton, M. (1991). Oogenesis, the formation of oogonial stalks and

fertilization in Sargassum vestitum (Fucales, Phaeophyta) from southern Australia.

Phycologia, 30(3), 243-256.

McCauley, J. I., Meyer, B. J., Winberg, P. C., Ranson, M., & Skropeta, D. (2015). Selecting

Australian marine macroalgae based on the fatty acid composition and anti-

inflammatory activity. Journal of Applied Phycology, 1-11.

Michalak, I., & Chojnacka, K. (2014). Algal extracts: Technology and advances.

Engineering in Life Sciences, 14(6), 581-591.

Mohamed, S., Hashim, S. N., & Rahman, H. A. (2012). Seaweeds: a sustainable functional

food for complementary and alternative therapy. Trends in Food Science & Technology,

23(2), 83-96.

Myers, J. H., Duda, S., Gunthorpe, L., & Allinson, G. (2007). Evaluation of the Hormosira

banksii (Turner) desicaine germination and growth inhibition bioassay for use as a

regulatory assay. Chemosphere, 69(6), 955-960.

Nakai, M., Kageyama, N., Nakahara, K., & Miki, W. (2006). Phlorotannins as radical

scavengers from the extract of Sargassum ringgoldianum. Marine Biotechnology, 8(4),

409-414.

Nakazawa, Y., Sashima, T., Hosokawa, M., & Miyashita, K. (2009). Comparative

evaluation of growth inhibitory effect of stereoisomers of fucoxanthin in human cancer

cell lines. Journal of Functional Foods, 1(1), 88-97.

Namvar, F., Mohamad, R., Baharara, J., Zafar-Balanejad, S., Fargahi, F., & Rahman, H. S.

(2013). Antioxidant, antiproliferative, and antiangiogenesis effects of polyphenol-rich

seaweed (Sargassum muticum). BioMed Research International, 2013.

http://dx.doi.org/10.1155/2013/604787

69

Ngo, D.H., Wijesekara, I., Vo, T.S., Van Ta, Q., & Kim, S.K. (2011). Marine food-derived

functional ingredients as potential antioxidants in the food industry: an overview. Food

Research International, 44(2), 523-529.

Nobili, S., Lippi, D., Witort, E., Donnini, M., Bausi, L., Mini, E., & Capaccioli, S. (2009).

Natural compounds for cancer treatment and prevention. Pharmacological Research,

59(6), 365-378.

Noviendri, D., Jaswir, I., Salleh, H. M., Taher, M., Miyashita, K., & Ramli, N. (2011).

Fucoxanthin extraction and fatty acid analysis of Sargassum binderi and S. duplicatum.

Journal of Medicial Plant Research, 11, 2405-2412.

Nwosu, F., Morris, J., Lund, V. A., Stewart, D., Ross, H. A., & McDougall, G. J. (2011).

Anti-proliferative and potential anti-diabetic effects of phenolic-rich extracts from

edible marine algae. Food Chemistry, 126(3), 1006-1012.

Nyiredy, S. (2004). Separation strategies of plant constituents–current status. Journal of

Chromatography B, 812(1-2), 35-51.

O’Sullivan, L., Murphy, B., McLoughlin, P., Duggan, P., Lawlor, P. G., Hughes, H., &

Gardiner, G. E. (2010). Prebiotics from marine macroalgae for human and animal

health applications. Marine Drugs, 8(7), 2038-2064.

Okada, T., Mizuno, Y., Sibayama, S., Hosokawa, M., & Miyashita, K. (2011). Antiobesity

effects of Undaria lipid capsules prepared with scallop phospholipids. Journal of Food

Science, 76(1).

Palanisamy, S., Vinosha, M., Marudhupandi, T., Rajasekar, P., & Prabhu, N. M. (2017).

Isolation of fucoidan from Sargassum polycystum brown algae: Structural

characterization, in vitro antioxidant and anticancer activity. International Journal of

Biological Macromolecules, 102, 405-412.

70

Pan, P.-S., Vasko, R. C., Lapera, S. A., Johnson, V. A., Sellers, R. P., Lin, C.-C., McAlpine,

S. R. (2009). A comprehensive study of Sansalvamide A derivatives: The structure–

activity relationships of 78 derivatives in two pancreatic cancer cell lines. Bioorganic &

Medicinal Chemistry, 17(16), 5806-5825.

Pangestuti, R., & Kim, S.K. (2011). Biological activities and health benefit effects of natural

pigments derived from marine algae. Journal of Functional Foods, 3(4), 255-266.

Pimentel-Elardo, S. M., Kozytska, S., Bugni, T. S., Ireland, C. M., Moll, H., & Hentschel,

U. (2010). Anti-parasitic compounds from Streptomyces sp. strains isolated from

Mediterranean sponges. Marine Drugs, 8(2), 373-380.

Polshettiwar, V., & Varma, R. S. (2008). Aqueous microwave chemistry: a clean and green

synthetic tool for rapid drug discovery. Chemical Society Reviews, 37(8), 1546-1557.

Pomin, V. H., & Mourão, P. A. (2008). Structure, biology, evolution, and medical

importance of sulfated fucans and galactans. Glycobiology, 18(12), 1016-1027.

Poore, A. G., Campbell, A. H., & Steinberg, P. D. (2009). Natural densities of mesograzers

fail to limit growth of macroalgae or their epiphytes in a temperate algal bed. Journal of

Ecology, 97(1), 164-175.

Poore, A. G., Watson, M. J., de Nys, R., Lowry, J. K., & Steinberg, P. D. (2000). Patterns of

host use among alga-and sponge-associated amphipods. Marine Ecology Progress

Series, 208, 183-196.

Qi, C., Gao, W., Guan, D., Wang, J., Liu, M., Chen, C., Hu, Z. (2018). Butenolides from a

Marine-Derived Fungus Aspergillus terreus with Antitumor Activities Against

Pancreatic Ductal Adenocarcinoma Cells. Bioorganic & Medicinal Chemistry.

Ragan, M. A., & Glombitza, K.W. (1986). Phlorotannins, brown algal polyphenols.

Progress in Phycological Research, 4, 129-241.

71

Rajauria, G., Foley, B., & Abu-Ghannam, N. (2016). Characterization of dietary

fucoxanthin from Himanthalia elongata brown seaweed. Food Research International,

99(3), 995-1001.

Ralph, P. J., Morrison, D. A., & Addison, A. (1998). A quantitative study of the patterns of

morphological variation within Hormosira banksii (Turner) Decaisne (Fucales:

Phaeophyta) in south-eastern Australia. Journal of Experimental Marine Biology and

Ecology, 225(2), 285-300.

Rauter, A. P., Martins, A., Borges, C., Ferreira, J., Justino, J., Bronze, M.R., Verpoorte, R.

(2005). Liquid chromatography–diode array detection–electrospray ionisation mass

spectrometry/nuclear magnetic resonance analyses of the anti-hyperglycemic flavonoid

extract of Genista tenera: Structure elucidation of a flavonoid-C-glycoside. Journal of

Chromatography A, 1089(1), 59-64.

Reddy, P., & Urban, S. (2009). Meroditerpenoids from the southern Australian marine

brown alga Sargassum fallax. Phytochemistry, 70(2), 250-255.

Rengarajan, T., Rajendran, P., Nandakumar, N., Balasubramanian, M. P., & Nishigaki, I.

(2013). Cancer preventive efficacy of marine carotenoid fucoxanthin: cell cycle arrest

and apoptosis. Nutrients, 5(12), 4978-4989.

Ritchie, R. J. (2006). Consistent sets of spectrophotometric chlorophyll equations for

acetone, methanol and ethanol solvents. Photosynthesis Research, 89(1), 27-41.

Roge, A., Firke, S., Kawade, R., Sarje, S., & Vadvalkar, S. (2011). Brief Review On: Flash

Chromatography. International Journal of Pharmaceutical Sciences and Research,

2(8), 1930.

5RPDQLN**LOJHQDVW(3U]\MD]Q\$ .DPLĔVNL0  7HFKQLTXHVRISUHSDULQJ

plant material for chromatographic separation and analysis. Journal of Biochemical and

Biophysical Methods, 70(2), 253-261. 72

Roselló-6RWR ( *DODQDNLV & 0 %UQþLü 0 2UOLHQ 9 7UXMLOOR ) - 0DZVRQ 5

Barba, F. J. (2015). Clean recovery of antioxidant compounds from plant foods, by-

products and algae assisted by ultrasounds processing. Modeling approaches to

optimize processing conditions. Trends in Food Science & Technology, 42(2), 134-149.

Sasidharan, S., Chen, Y., Saravanan, D., Sundram, K., & Latha, L. Y. (2011). Extraction,

isolation and characterization of bioactive compounds from plants’ extracts. African

Journal of Traditional, Complementary and Alternative Medicines, 8(1).

Senthilkumar, K., Manivasagan, P., Venkatesan, J., & Kim, S.-K. (2013). Brown seaweed

fucoidan: Biological activity and apoptosis, growth signaling mechanism in cancer.

International Journal of Biological Macromolecules, 60, 366-374.

Sforza, S., Dall'Asta, C., & Marchelli, R. (2006). Recent advances in mycotoxin

determination in food and feed by hyphenated chromatographic techniques/mass

spectrometry. Mass Spectrometry Reviews, 25(1), 54-76.

Sharma, S. H., Thulasingam, S., & Nagarajan, S. (2016). Terpenoids as anti-colon cancer

agents–A comprehensive review on its mechanistic perspectives. European Journal of

Pharmacology.

Sherma, J. (2000). Thin-layer chromatography in food and agricultural analysis. Journal of

Chromatography A, 880(1-2), 129-147.

Shi, D., Li, J., Guo, S., Su, H., & Fan, X. (2009). The antitumor effect of bromophenol

derivatives in vitro and Leathesia nana extract in vivo. Chinese Journal of Oceanology

and Limnology, 27, 277-282.

Shirsath, S., Sonawane, S., & Gogate, P. (2012). Intensification of extraction of natural

products using ultrasonic irradiations—a review of current status. Chemical

Engineering and Processing: Process Intensification, 53, 10-23.

73

Singh, I. P., & Bharate, S. B. (2006). Phloroglucinol compounds of natural origin. Natural

Product Reports, 23(4), 558-591.

Steinberg, P. D. (1994). Lack of short-term induction of phlorotannins in the Australasian

brown algae Ecklonia radiata and Sargassum vestitum. Marine Ecology Progress

Series 112, 129-133.

Sticher, O. (2008). Natural product isolation. Natural Product Reports, 25(3), 517-554.

Sugiura, Y., Matsuda, K., Yamada, Y., Nishikawa, M., Shioya, K., Katsuzaki, H., AMANO,

H. (2007). Anti-allergic phlorotannins from the edible brown alga, Eisenia arborea.

Food Science and Technology Research, 13(1), 54-60.

Tanaka, T., Shnimizu, M., & Moriwaki, H. (2012). Cancer chemoprevention by carotenoids.

Molecules, 17(3), 3202-3242.

Targett, N. M., & Arnold, T. M. (1998). Minireview—predicting the effects of brown algal

phlorotannins on marine herbivores in tropical and temperate oceans. Journal of

Phycology, 34(2), 195-205.

Taylor, L. T. (2009). Supercritical fluid chromatography for the 21st century. The Journal of

Supercritical Fluids, 47(3), 566-573.

Teh, S.S., & Birch, E. J. (2014). Effect of ultrasonic treatment on the polyphenol content

and antioxidant capacity of extract from defatted hemp, flax and canola seed cakes.

Ultrasonics Sonochemistry, 21(1), 346-353.

Thomas, N. V., & Kim, S.-K. (2011). Potential pharmacological applications of

polyphenolic derivatives from marine brown algae. Environmental Toxicology and

Pharmacology, 32(3), 325-335.

Tsao, R., & Deng, Z. (2004). Separation procedures for naturally occurring antioxidant

phytochemicals. Journal of Chromatography B, 812(1), 85-99.

74

Underwood, A. (1998). Grazing and disturbance: an experimental analysis of patchiness in

recovery from a severe storm by the intertidal alga Hormosira banksii on rocky shores

in New South Wales. Journal of Experimental Marine Biology and Ecology, 231(2),

291-306.

Valentine, J. P., & Johnson, C. R. (2004). Establishment of the introduced kelp Undaria

pinnatifida following dieback of the native macroalga Phyllospora comosa in

Tasmania, Australia. Marine and Freshwater Research, 55(3), 223-230.

Verbeke, C., Löhr, M., Karlsson, J. S., & Del Chiaro, M. (2015). Pathology reporting of

pancreatic cancer following neoadjuvant therapy: challenges and uncertainties. Cancer

Treatment Reviews, 41(1), 17-26.

Vilkhu, K., Mawson, R., Simons, L., & Bates, D. (2008). Applications and opportunities for

ultrasound assisted extraction in the food industry—A review. Innovative Food Science

& Emerging Technologies, 9(2), 161-169.

Vuong, D., Kaplan, M., Lacey, H. J., Crombie, A., Lacey, E., & Piggott, A. M. (2018). A

study of the chemical diversity of macroalgae from South Eastern Australia.

Fitoterapia, 126, 53-64.

Wang, J., Sun, B., Cao, Y., Tian, Y., & Li, X. (2008). Optimisation of ultrasound-assisted

extraction of phenolic compounds from wheat bran. Food Chemistry, 106(2), 804-810.

Wang, L., & Weller, C. L. (2006). Recent advances in extraction of nutraceuticals from

plants. Trends in Food Science & Technology, 17(6), 300-312.

Wang, T., Jónsdóttir, R. s., Liu, H., Gu, L., Kristinsson, H. G., Raghavan, S., & Ólafsdóttir,

G. n. (2012). Antioxidant capacities of phlorotannins extracted from the brown algae

Fucus vesiculosus. Journal of Agricultural and Food Chemistry, 60(23), 5874-5883.

75

Wijesekara, I., & Kim, S.-K. (2010). Angiotensin-I-converting enzyme (ACE) inhibitors

from marine resources: prospects in the pharmaceutical industry. Marine Drugs, 8(4),

1080-1093.

Wijesinghe, W., & Jeon, Y.-J. (2012). Biological activities and potential industrial

applications of fucose rich sulfated polysaccharides and fucoidans isolated from brown

seaweeds: A review. Carbohydrate Polymers, 88(1), 13-20.

Wong, K., & Cheung, P. C. (2001). Influence of drying treatment on three Sargassum

species. Journal of Applied Phycology, 13(1), 43-50.

Wu, L., Sun, J., Su, X., Yu, Q., Yu, Q., & Zhang, P. (2016). A review about the

development of fucoidan in antitumor activity: Progress and challenges. Carbohydrate

Polymers, 154, 96-111.

Xiao, W., Han, L., & Shi, B. (2008). Microwave-assisted extraction of flavonoids from

Radix Astragali. Separation and Purification Technology, 62(3), 614-618.

Xu, Q., Zhang, T.-P., & Zhao, Y.-P. (2011). Advances in early diagnosis and therapy of

pancreatic cancer. Hepatobiliary & Pancreatic Diseases International, 10(2), 128-135.

Xue, M., Ge, Y., Zhang, J., Wang, Q., Hou, L., Liu, Y., Li, Q. (2012). Anticancer properties

and mechanisms of fucoidan on mouse breast cancer in vitro and in vivo. PloS one,

7(8), e43483.

Yamasaki-Miyamoto, Y., Yamasaki, M., Tachibana, H., & Yamada, K. (2009). Fucoidan

induces apoptosis through activation of caspase-8 on human breast cancer MCF-7 cells.

Journal of Agricultural and Food Chemistry, 57(18), 8677-8682.

Yan, X., Chuda, Y., Suzuki, M., & Nagata, T. (1999). Fucoxanthin as the major antioxidant

in Hijikia fusiformis, a common edible seaweed. Bioscience, biotechnology, and

biochemistry, 63(3), 605-607.

76

Yang, C., Chung, D., Shin, I.S., Lee, H., Kim, J., Lee, Y., & You, S. (2008a). Effects of

molecular weight and hydrolysis conditions on anticancer activity of fucoidans from

sporophyll of Undaria pinnatifida. International Journal of Biological

Macromolecules, 43(5), 433-437.

Yang, C., Chung, D., & You, S. (2008b). Determination of physicochemical properties of

sulphated fucans from sporophyll of Undaria pinnatifida using light scattering

technique. Food Chemistry, 111(2), 503-507.

Yang, H., Zeng, M., Dong, S., Liu, Z., & Li, R. (2010). Anti-proliferative activity of

phlorotannin extracts from brown algae Laminaria japonica Aresch. Chinese Journal of

Oceanology and Limnology, 28, 122-130.

Yang, L., Wang, P., Wang, H., Li, Q., Teng, H., Liu, Z., Zou, X. (2013). Fucoidan derived

from Undaria pinnatifida induces apoptosis in human hepatocellular carcinoma

SMMC-7721 cells via the ROS-mediated mitochondrial pathway. Marine Drugs, 11(6),

1961-1976.

Zhang, Z., Wang, F., Wang, X., Liu, X., Hou, Y., & Zhang, Q. (2010). Extraction of the

polysaccharides from five algae and their potential antioxidant activity in vitro.

Carbohydrate polymers, 82(1), 118-121.

Ye, H., Wang, K., Zhou, C., Liu, J., & Zeng, X. (2008). Purification, antitumor and

antioxidant activities in vitro of polysaccharides from the brown seaweed Sargassum

pallidum. Food Chemistry, 111(2), 428-432.

Ye, M., Han, J., Chen, H., Zheng, J., & Guo, D. (2007). Analysis of phenolic compounds in

rhubarbs using liquid chromatography coupled with electrospray ionization mass

spectrometry. Journal of the American Society for Mass Spectrometry, 18(1), 82-91.

Yoshida, T. (1997). Peptide separation in normal phase liquid chromatography. Analytical

Chemistry, 69(15), 3038-3043. 77

Yoshida, T. (2004). Peptide separation by hydrophilic-interaction chromatography: a

review. Journal of Biochemical and Biophysical Methods, 60(3), 265-280.

You, S., Yang, C., Lee, H., & Lee, B.Y. (2010). Molecular characteristics of partially

hydrolyzed fucoidans from sporophyll of Undaria Pinnatifida and their in vitro

anticancer activity. Food Chemistry, 119(2), 554-559.

Yu, R.X., Hu, X.M., Xu, S.Q., Jiang, Z.J., & Yang, W. (2011). Effects of fucoxanthin on

proliferation and apoptosis in human gastric adenocarcinoma MGC-803 cells via

JAK/STAT signal pathway. European Journal of Pharmacology, 657(1-3), 10-19.

Zhang, H.-F., Yang, X.-H., & Wang, Y. (2011). Microwave assisted extraction of secondary

metabolites from plants: current status and future directions. Trends in Food Science &

Technology, 22(12), 672-688.

Zhang, Q., Yu, P., Li, Z., Zhang, H., Xu, Z., & Li, P. (2003). Antioxidant activities of

sulfated polysaccharide fractions from Porphyra haitanesis. Journal of Applied

Phycology, 15(4), 305-310.

Zhang, Z., Zhang, P., Hamada, M., Takahashi, S., Xing, G., Liu, J., & Sugiura, N. (2008).

Potential chemoprevention effect of dietary fucoxanthin on urinary bladder cancer EJ-1

cell line. Oncology Reports, 20(5), 1099-1103.

Zhou, H.-Y., & Liu, C.Z. (2006). Microwave-assisted extraction of solanesol from tobacco

leaves. Journal of Chromatography A, 1129(1), 135-139.

78