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Title Cellular and in-vitro models to assess antioxidant activities of seaweed
extracts and the potential use of the extracts as ingredients
Author(s) O'Sullivan, Anthoney Michael Naim
Publication date 2013
Original citation O'Sullivan, A.M.N. 2013. Cellular and in-vitro models to assess
antioxidant activities of seaweed extracts and the potential use of the
extracts as ingredients. PhD Thesis, University College Cork.
Type of publication Doctoral thesis
Rights © 2013, Anthoney M. N. O'Sullivan
http://creativecommons.org/licenses/by-nc-nd/3.0/
Embargo information No embargo required
Item downloaded http://hdl.handle.net/10468/1234
from
Downloaded on 2023-01-14T01:08:16Z
The National University of Ireland
Cork
School of Food and Nutritional Sciences
Supervisors: Professor N.M. O’Brien and Dr. J.P. Kerry

Cellular and In-vitro Models to Assess Antioxidant
Activities of Seaweed Extracts and the Potential Use of the
Extracts as Ingredients

Thesis presented by
Anthoney Michael Naim O’Sullivan, B.Sc.

For the degree of
Doctor of Philosophy in Food and Nutritional Sciences

January, 2013
i
Table of Contents
Acknowledgements iii
Publications v
Abstract ix

Chapter 1 1
Literature review

Chapter 2 57
Antioxidant activities assessed using in-vitro and cellular model systems
of five brown seaweeds harvested in spring from the west coast of Ireland.

Chapter 3 85
Assessment of the ability of seaweed extracts to protect against hydrogen
peroxide and tert-butyl hydroperoxide induced cellular damage in Caco-2 cells.

Chapter 4 101
The effect of solvents on the antioxidant activity in Caco-2 cells of Irish
brown seaweed extracts prepared using accelerated solvent extraction (ASE®).

Chapter 5 125
An examination of the potential of seaweed extracts as functional ingredients
in milk.

Chapter 6 162
An investigation of the performance of seaweed extracts as potential functional
ingredients in yogurt.

Chapter 7 204
Additional study: Assessment of the antioxidant potential of extracts of
Chlorella, Spirulina and Kelp food supplements in Caco-2 cells.

Chapter 8 225
General discussion

ii
Acknowledgements
First and foremost I want to sincerely thank my supervisor Dr. Joseph Kerry and my
funding authority Nutramara for giving me this great opportunity to pursue my PhD.
Joe is a great man and a man of his word.
Next I would like to take this opportunity to thank my co-supervisor Professor Nora
O’Brien. Her unbounded help and inspiration has been instrumental in the success
of my PhD. She is always there to give her students the advice and guidance they
need. Even if she is away on business on the other side of the world she is just an
email away at all times. This gives all her students the peace of mind they require
when potential problems may arise. I cannot put in words how much I appreciate
Nora, for the corrections and suggestions she gave me on various topics of my PhD
whether it be a paper, conference abstract, poster or PowerPoint presentation. Her
input often made something that was ordinary into something excellent. I could type
pages on Nora but I think I have said enough.
Now I would like to thank Yvonne O’Callaghan, my assigned research officer,
whose advice, help and guidance made my PhD journey a very fruitful, educational
and happy one. She is always there to help whether it is lab work or paperwork such
as research papers, conference abstracts, posters or PowerPoint presentations. Just
like I said about Nora, Yvonne is always just an email/text away in case I need help.
Next I would like to thank Mike O’Grady, my other assigned research officer, for all
his help with setting up experiments and correcting my papers. I would like to thank
Mike for all the support he has given me in the final stages of my thesis writing.
The technical staff in Nutrition is the best I have ever met. Bernice Quinn and Dr.
Karen Galvin are always there to help me (or anyone for that matter) at the drop of a
hat. There is no such thing as “call back tomorrow” or “can you call sometime next
week instead?” as BQ and KG will make you wait a few minutes at most.
Three very similar people are Jim Holland, Paddy O’Reilly and Eddie Beattie MSc.
Jim and Eddie have never said no to me whenever I needed help regardless of how
busy they are themselves. Eddie is an utter gentlemen and one of the nicest people I
have ever met. Without Paddy I would have no clue about what to do when it came
to microbiology e.g. what agars to use, what incubators to use etc. Laurie O’Sullivan
you are a great friend and a great support even since you left UCC. If I’m ever a
little puzzled with some lab work question I just give Laurie a text or email and she
sorts me out within a few hours.
iii
While doing my PhD I was given the opportunity to lecture once a year and I would
like to sincerely thank Professor Nora O’Brien and Professor Kevin Cashman for
allowing me to do this. In addition I was given the opportunity to tutor in food and
nutritional science subjects. I would like to thank Dr. Tom O’Connor, Dr. Joseph
Kerry, Professor Yrjo Roos, Professor Paul McSweeney, and Eddie Beattie (again)
for helping me formulate the tutorial notes I required. Thanks to Dave Waldron for
helping me with the yogurt and milk trials.
Thanks to Dr. Brian Tobin for his help with analysing my sensory data etc.
Thanks to Pat O’Connell, Dr. Tom O’Connor and Dr. Tony Sheehy for their support
and advice.
During my BSc I had some excellent lectures from some excellent lecturers which
gave me the inspiration to try and pursue a PhD in Food/ Nutritional Sciences. The
excellent lecturers I speak of are Dr. Joseph Kerry, Professor Nora O’Brien, Dr. Tom
O’Connor, Professor Gerald Fitzgerald, Professor Colin Hill and Professor Tom
Cotter. I would also like to thank some old pre-college teachers including Mr. J.J.
Murphy, Mr. Brendan Nally, Miss Anne Kelly, Miss Susan Buckley, and Miss
Michelle McAdoo who always believed in me and gave me the determination to
study and work hard in school. I would like to thank my college friends for their
support throughout my BSc and in life in general and for all the great memories
including the G’pino Fraternity (Kev Murphy, Noely McCarthy, Diarmo Mullins,
Rony Murphy and Ms. Aida Whooley), PJ Fox, Steve Daly, Maria McKenna, and
Ber Kirby. Also I would like to thank the friends I made during the PhD for all their
support and help. These include Marvin Jiwan, Olivia Kenny, Aoife McCarthy,
Martha Phelan and Trevor Daly. Also I would like to thank my lifelong friends
Liam and Ted for their support when needed. Great thanks to Mrs. Murphy for all
her kindness to me.
Finally I would like to thank my family for all their support throughout the years
especially my best friend/ Nana Mary, my Granda John, mother Helena, uncles
Michael and Johnny, aunts Mag and Maura, cousin/best friend Joe, Alexandra, Jack
and all the rest of my family and God. My Nana, Granda and Mother were very
supportive (especially financially) in all aspects of my education from my days in
Montessori school right down to my PhD days. Without their constant support I
would not be where I am now.
Thank you to all.
iv
Publications

Papers:
1. O’Sullivan, A.M., O’Callaghan, Y.C., O’Grady, M.N., Queguineur, B.,
Hanniffy, D., Troy, D.J., Kerry, J.P. & O’Brien, N.M. (2011). In-vitro and
cellular antioxidant activities of seaweed extracts prepared from five brown
seaweeds harvested in spring from the west coast of Ireland. Food Chemistry,
126, 1064-1070.
2. O’Sullivan, A.M., O’Callaghan, Y.C., O’Grady, M.N., Queguineur, B.,
Hanniffy, D., Troy, D.J., Kerry, J.P. & O’Brien, N.M. (2012). Assessment of the
ability of seaweed extracts to protect against hydrogen peroxide and tert-butyl
hydroperoxide induced cellular damage in Caco-2 cells. Food Chemistry, 134,
1137-1140.
3. O’Sullivan, A.M., O’Callaghan, Y.C., O’Grady, M.N., Hayes, M., Kerry, J.P. &
O’Brien, N.M. (2013). Use of a Caco-2 cell model to assess the antioxidant
activity of Irish brown seaweed extracts prepared using accelerated solvent
extraction (ASE®). Journal of Functional Foods. (DOI number:
10.1016/j.jff.2013.02.007).
4. O’Sullivan, A.M., O’Callaghan, Y.C., O’Connor, T.P. & O’Brien, N.M. (2013).
Comparison of the antioxidant activity of commercial honeys, before and after
in-vitro digestion. Polish Journal of Food and Nutrition Sciences. (DOI
number: 10.2478/v10222-012-0080-6).
5. O’Sullivan, A.M., O’Callaghan, Y.C., O’Grady, M.N., Smyth, T., O’Brien, N.M,
& Kerry, J.P. (2013). An Examination of the Potential of Seaweed Extracts as
Functional Ingredients in Milk. International Journal of Dairy Technology.
(Submitted).
v
6. O’Sullivan, A.M., O’Grady, M.N., O’Callaghan, Y.C., Smyth, T., O’Brien, N.M,
& Kerry, J.P. (2013). Seaweed Extracts as Potential Functional Ingredients in
Yogurt. LWT- Food Science and Technology. (Submitted).

vi
Abstracts
1. O’Sullivan, A.M., O’Callaghan, Y.C., O’Grady, M.N., Kraan, S., Kerry, J.P. &
O’Brien, N.M. (2009). Determination of the antioxidant potential of seaweed
extracts using different methods. Proceedings of the Nutrition Society, 68,
OCE3, E110.
2. O’Sullivan, A.M., O’Callaghan, Y.C., O’Grady, M.N., Kerry, J.P. & O’Brien,
N.M. (2010). The antioxidant activity of five brown seaweeds, sourced from the
west coast of Ireland, assessed in the Caco-2 cell line. Proceedings of the
Nutrition Society, 69, OCE6, E403.
3. O’Sullivan, A.M., O’Callaghan, Y.C., O'Connor, T. P, & O’Brien, N.M. (2011).
The content and bioaccessibility of carotenoids from selected commercially
available health supplements. Proceedings of the Nutrition Society, 70, OCE3,
E62.
4. O’Sullivan, A.M., O’Callaghan, Y.C., O’Grady, M.N., Hayes, M., Kerry, J.P. &
O’Brien, N.M. (2010). The influence of the extraction method on the DNA
protective effects of seaweed extracts in Caco-2 cells. Proceedings of the
Nutrition Society, 70, OCE3, E77.
5. O’Sullivan, A.M., O’Callaghan, Y.C., O'Connor, T. P, & O’Brien, N.M. (2011).
Bioaccessibility of polyphenols and carotenoids from wild Irish berries subjected
to an in-vitro digestion procedure. Proceedings of the Nutrition Society, 70,
OCE3, E61.
6. O’Sullivan, A.M., O’Callaghan, Y.C., O’Grady, M.N., Queguineur, B.,
Hanniffy, D., Troy, D.J., Kerry, J.P. & O’Brien, N.M. (2011).The ability of
seaweed extracts to protect against oxidative damage to DNA in Caco-2 cells.
Proceedings of the 40th Annual UCC Food Research Conference, A31.
vii
7. O’Sullivan, A.M., O’Callaghan, Y.C., O’Grady, M.N., Hayes, M., Kerry, J.P. &
O’Brien, N.M. (2012). The DNA-protective effect of seaweed extracts is
dependent on the extraction solvent. Proceedings of the 41st Annual UCC Food
Research Conference, A1.
8. O’Callaghan, Y.C., O’Sullivan, A.M., O'Connor, T. P, & O’Brien, N.M. (2012).
The effect of an in-vitro digestion on the antioxidant activity of a range of
honeys. Proceedings of the 41st Annual UCC Food Research Conference, P12.
viii
Abstract
Seaweeds contain a range of antioxidant compounds such as polyphenols,
carotenoids, sulphated polysaccharides and vitamins and have the potential to be
used as ingredients in neutraceuticals. The antioxidant activity of crude 60%
methanol extracts prepared from five Irish seaweeds, Ascophyllum nodosum,
Laminaria hyperborea, Pelvetia canaliculata, Fucus vesiculosus and Fucus serratus
were examined using in-vitro assays and a cell model system to determine the
antioxidant activity of the extracts and their ability to protect against H O and tert-
2 2
BOOH-induced DNA damage and alterations in cellular antioxidant status in the
human adenocarcinoma, Caco-2 cell line.
To optimise the extraction of antioxidant compounds from seaweeds, an
accelerated solvent extraction (ASE®) was used in combination with food grade
solvents. The antioxidant activity of these extracts against H O and tert-BOOH-
2 2
induced DNA damage and alterations in cellular antioxidant status was also assessed.
Extracts that exhibited the highest antioxidant activity, A. nodosum (100%
water and 80% ethanol extracts) and F. vesiculosus (60% ethanol extract) were
selected as ingredients for incorporation into fluid milk and yogurt at concentrations
of 0.25% and 0.5%. The addition of the seaweed extracts to milk and yogurt did not
affect the pH or shelf-life properties of the products. Seaweed addition did however
significantly influence the colour properties of the milk and yogurt. Yellowness
values were significantly higher in yogurts containing F. vesiculosus at both

concentrationsand A. nodosum (80% ethanol)at the 0.5% concentration. In milk, the

F. vesiculosus (60% ethanol) and A. nodosum (80% ethanol) at both the 0.25% and

the 0.5% concentrations had higher greenness and yellowness values than the milk
containing A. nodosum (100% water).
Sensory analysis revealed that appearance and flavour governed the overall
acceptability of yogurts with the control yogurt, and yogurts containing A. nodosum
(100% water) were the most preferred samples by panellists. However, in the milk
trial the perception of a fishy taste was the determining factor in the negative
perception of milk. The unsupplemented control and the milk containing A.
nodosum (100% water) at a concentration of 0.5% were the most overall accepted
milk samples by the sensory panellists.
The antioxidant activity of the extracts in milk and yogurt remained stable
during storage as determined by the in-vitro assays. Seaweed supplemented milk
ix