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 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 Paper I: 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) J Food Process Pres 41(4): e13025 doi.org/10.1111/jfpp.13025 Paper II: Dang TT, Bowyer MC, Van Altena IA & Scarlett CJ (2018) Comparison of chemical profile and antioxidant properties of the brown algae, Inter Food Sci Technol 51(1): 174-181 doi: 10.1111/ijfs.13571 Paper III: Dang TT, Vuong QV, Schreider MJ, Bowyer MC, Van Altena IA & Scarlett CJ (2017) Optimisation of ultrasound-assisted extraction conditions for phenolic content and antioxidant activities of the alga Hormosira banksii using response surface methodology J Appl Phycol 29(6): 3161-3173 doi.org/10.1007/s10811-017-1162-y 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 Sep Sci Technol (In Press) doi.org/10.1080/01496395.2017.1414845 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 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 Paper VII: Dang TT, Sakoff JA, Bowyer MC, Van Altena IA & Scarlett CJ Antioxidant and cytotoxic activity (in vitro) of phlorotannin-enriched fractions from the brown alga Hormosira banksii (Turner) Decaisne Submitted to Journal of MarineBiotechnology Paper VIII: Dang TT & Scarlett CJ Extraction and cytotoxic activity of the sulfated polysaccharides (fucoidans) against pancreatic cancer in vitro from brown alga Hormosira banksii (Turner) Decaisne Submitted to Journal of Biomedicine andPharmacotherapy v STATEMENT OF AUTHORS' CONTRIBUTION TO THE PAPERS To whom it may concern, We, Quan V Vuong, Maria J Schreider, Christopher J Scarlett, Michael C Bowyer and Ian A Van Altena as co-authors with contribution of planning, giving the suggestions and editing the paper, attest that research higher degree candidate, Thanh Trung Dang, was the principle contributor to the planning, execution, analyses of the experiments and the writing of the published research paper entitled “The Effects of Drying on Physico‐Chemical Properties and Antioxidant Capacity of the Brown Alga (Hormosira banksii (Turner) Decaisne)” J Food Process Pres 41(4): e13025 doi.org/10.1111/jfpp.13025 Dr Ian A Van Altena 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, Christopher J Scarlett, Michael C Bowyer and Ian A Van Altena as co-authors with contribution of planning, giving the giving the suggestions and editing the paper, attest that research higher degree candidate, Thanh Trung Dang, was the principle contributor to the planning, execution, analyses of the experiments and the writing of the published research paper entitled “Comparisonof chemical profileandantioxidantproperties of the brown algae”, Inter Food Sci Technol 51(1): 174-181 doi:10.1111/ijfs.13571 Dr Ian A Van Altena 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: x STATEMENT OF AUTHORS' CONTRIBUTION TO THE PAPERS To whom it may concern, We, Quan V Vuong, Maria J Schreider, Christopher J Scarlett, Michael C Bowyer and Ian A Van Altena as co-authors with contribution of planning, giving the giving the suggestions and editing the paper, attest that research higher degree candidate, Thanh Trung Dang, was the principle contributor to the planning, execution, analyses of the experiments and the writing of the published research paper entitled “Optimisation of ultrasound-assisted extraction conditions for phenolic content and antioxidant activities of the alga Hormosira banksii using response surface methodology” J Appl Phycol 29(6): 31613173 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: xi 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” Sep Sci Technol (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: xii 2015) Until now, little information about the cytotoxic effects of fucoxanthin against pancreatic cancer cells has been reported 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) The phlorotannin-enriched fractions from the algae with the high cancer cell growth inhibition have been outlined previously The fraction with a high ratio of phenolics from the Ecklonia cava extract exhibited high inhibitory activity on the murine colon cancer cell line (CT-26) and mouse melanoma cell line (B-16) with the values of IC50= 5.1 and 29.3 µg.mL-1, respectively, with low toxicity to the normal cells (V79-4) with the inhibition < 20% at the dosages of 5-100 µg.mL-1 (Athukorala et al., 2006) There was a correlation of phenolics from Laminaria japonica Aresch with the inhibitory effects against the human hepatocellular carcinoma cell (BEL-7402) line and on murine leukemic cells (P388) (Yang et al, 2010) In addition, the Alaria esculenta extract with a low phenolics level showed high growth inhibition towards the colon cancer cell (Caco-2) (Nwosu et al., 2011) The hexane fractions from Sargassum swartzii and Cystoseira myrica showed selective cytotoxicity against proliferation of colon adenocarcinoma and breast carcinoma, while they demonstrated low toxicity to the normal cells NIH 3T3 (Swiss embryo fibroblast) with IC50 values of 180.7 and 639.74 µg.mL-1, respectively (Khanavi et al., 2010) Therefore, phenolics from the different algal species resulted in the varying inhibitory efficacy across numerous cancer cell lines 42 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 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 3.2 Conclusions and recommendations 3.2.1 Conclusions In the project, samples of six brown algae (Sargassum vestitum; Sargassum linearifolium; Phyllospora comosa; Padina sp.; Hormosira banksii; Sargassum podocanthum) were 43 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 For drying samples, the three methods of vacuum, de-humidification and freeze drying were suitable for the preparation of algal samples (H.banksii, Paper I) Three out of the six species (Sargassum vestitum; Padina sp and Hormosira banksia) possessed higher yield of constituents with high antioxidant activities compared to the remaining species, and were comparable with synthetic antioxidants These species were chosen for further study (Paper II) 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, and for S.vestitum in Paper IV The findings from Paper V showed that isolation of phenolic compounds from the H.banksii was efficient by the solvent-partitioning technique Higher yield of phenolics and antioxidant activity were found in the ethyl acetate and butanol fractions compared to other fractions, and the crude extract 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 was isolated via column chromatography and quantitated by 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 acetate fraction significantly inhibited numerous 44 cancer cells types (inhibition efficacies ≥ 80% with all cell lines) For pancreatic cancer, both the ethyl acetate and butanol fractions 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 3.2.2 Recommendations Based on the results obtained from this study, the author recommends the following studies on brown algae: • The polar phenolic compounds of the butanol fraction from alga H.banksii should be isolated and assessed for activities against pancreatic cancer cell lines • Fucoidans from fucoidan enriched fractions from alga H.banksii should be purified to enhance their observed activities against pancreatic cancer cell lines • Phenolic compounds from alga S.vestitum should be assessed for activities against pancreatic cancer cell lines • 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 • Fucoxanthin, phenolic compounds and fucoidan should be assessed for their mechanisms of action (apoptosis/cell cycle) against pancreatic cancer cell lines 45 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 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 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Chromatographic separation and isolation ofbioactive compounds Identification of bioactive compounds Activities of algal compounds against several cancer cell lines Algal components against pancreatic. .. 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. .. 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