Extraction, Characterization and Antioxidant Activity of Fucoidan from New Zealand Undaria ...

104 Pages·2012·1.86 MB·English

Extraction, Characterization and Antioxidant Activity of Fucoidan
from New Zealand Undaria pinnatifida (Harvey) Suringar

by

Wilfred Wing Fung Mak

A Thesis
Submitted to the Faculty of Health and Environmental Sciences of
Auckland University of Technology
in Partial Fulfillment of the Requirements
for the Degree of

MASTER OF APPLIED SCIENCE (MAppSc)

Auckland, New Zealand
March 2012
Extraction, Characterization and Antioxidant Activity of Fucoidan
from New Zealand Undaria pinnatifida (Harvey) Suringar

Approved by:
Advisors:
Dr. Lindsey White

Dr. Nazimah Hamid

Date approved:

Table of Contents

TABLE OF CONTENTS ................................................................................................ I
LIST OF FIGURES ....................................................................................................... V
LIST OF TABLES ...................................................................................................... VII
ATTESTATION OF AUTHORSHIP ..................................................................... VIII
DEDICATION .............................................................................................................. IX
ACKNOWLEDGEMENTS ........................................................................................... X
ABSTRACT ................................................................................................................. XII
1 INTRODUCTION ........................................................................................................ 1
1.1 Problem background .............................................................................................. 1
1.2 Purpose of the study ............................................................................................... 2
1.3 Overview ................................................................................................................ 3
2 REVIEW OF THE LITERATURE ............................................................................ 4
2.1 Introduction ............................................................................................................ 4
2.1.1 Biology of U. pinnatifida ............................................................................................................ 5
2.1.2 Distribution of U. pinnatifida in New Zealand ........................................................................... 6
2.1.3 Impact of U. pinnatifida in New Zealand ................................................................................... 8
2.2 Management of U. pinnatifida in New Zealand..................................................... 9
2.2.1 Management history ................................................................................................................... 9
2.2.2 Current management ................................................................................................................. 10
2.3 Previous research in New Zealand ....................................................................... 11
2.3.1 Department of Conservation (DoC) .......................................................................................... 11
2.3.2 Cawthron Institute .................................................................................................................... 12
2.3.3 Ministry of Fisheries (MFish) ................................................................................................... 12
2.3.4 Universities ............................................................................................................................... 13
2.4 Fucoidan: A sulphated fucan ................................................................................ 13
2.4.1 Introduction .............................................................................................................................. 13
2.4.2 Structure.................................................................................................................................... 14
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2.4.3 Extracting fucoidan ................................................................................................................... 15
2.4.4 Health benefits of fucoidan ....................................................................................................... 17
2.4.4.1 Anticoagulant activity ...................................................................................................... 20
2.4.4.2 Anticancer activity ........................................................................................................... 21
2.4.4.3 Antioxidant activity .......................................................................................................... 22
2.4.5 Variations in fucoidan composition .......................................................................................... 24
3 COMPARISON OF THREE EXTRACTION TECHNIQUES OF FUCOIDAN 26
3.1 Introduction .......................................................................................................... 26
3.2 Methods ................................................................................................................ 26
3.2.1 Harvesting ................................................................................................................................. 26
3.2.2 Drying ....................................................................................................................................... 27
3.2.3 Fucoidan extraction .................................................................................................................. 28
3.2.3.1 Method I - Water extraction ............................................................................................. 28
3.2.3.2 Method II – Acid extraction ............................................................................................. 28
3.2.3.3 Method III – Salt extraction ............................................................................................. 29
3.2.4 Fucoidan composition ............................................................................................................... 30
3.2.4.1 Fucose content ................................................................................................................. 30
3.2.4.2 Sulphate content ............................................................................................................... 30
3.2.4.3 Uronic acid content .......................................................................................................... 31
3.2.4.4 Protein content ................................................................................................................. 31
3.2.5 Statistical analysis ..................................................................................................................... 32
3.3 Results .................................................................................................................. 32
3.3.1 Extraction.................................................................................................................................. 32
3.3.2 Constituents of crude fucoidan ................................................................................................. 34
3.4 Discussion ............................................................................................................ 35
3.5 Conclusion ........................................................................................................... 37
4 MONTHLY CHANGES IN FUCOIDAN CONTENT AND COMPOSITION ... 39
4.1 Introduction .......................................................................................................... 39
4.2 Materials and methods ......................................................................................... 39
4.2.1 Sample collection and drying ................................................................................................... 39
4.2.2 Fucoidan extraction .................................................................................................................. 40
4.2.3 Fucoidan composition ............................................................................................................... 41
4.2.4 Statistical analysis ..................................................................................................................... 42
4.3 Results .................................................................................................................. 42
4.3.1 Monthly changes in crude fucoidan content and composition ...................................................... 42
4.3.2 Differences in crude fucoidan content between farms .............................................................. 46
4.4 Discussion ............................................................................................................ 46
4.5 Conclusion ........................................................................................................... 50
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5 ANTIOXIDANT ACTIVITY OF FUCOIDAN ....................................................... 51
5.1 Introduction .......................................................................................................... 51
5.2 Materials and methods ......................................................................................... 51
5.2.1 Sample collection...................................................................................................................... 51
5.2.2 Fucoidan extraction .................................................................................................................. 51
5.2.3 Fractionation of fucoidan .......................................................................................................... 52
5.2.3.1 Swelling the gel ................................................................................................................ 52
5.2.3.2 Packing and washing the column ..................................................................................... 52
5.2.3.3 Fractionation of fucoidan for analysis ............................................................................. 53
5.2.4 Fucoidan fraction composition ................................................................................................. 54
5.2.5 Antioxidant activity .................................................................................................................. 54
5.2.5.1 DPPH free-radical scavenging activity ........................................................................... 54
5.2.5.2 CUPRAC Assay ................................................................................................................ 55
5.2.6 Molecular weight determination ............................................................................................... 55
5.2.7 Statistical analysis ..................................................................................................................... 56
5.3 Results .................................................................................................................. 57
5.3.1 Fractionation and molecular weight determination of fucoidan ............................................... 57
5.3.2 Antioxidant activity .................................................................................................................. 59
5.4 Discussion ............................................................................................................ 61
5.5 Conclusion ........................................................................................................... 65
6 ADDITIONAL EXPERIMENTS ............................................................................. 66
6.1 Fucoidan in commercial seaweed ........................................................................ 66
6.1.1 Introduction .............................................................................................................................. 66
6.1.2 Materials and methods .............................................................................................................. 66
6.1.3 Results and discussion .............................................................................................................. 67
6.2 Re-extracting fucoidan from U. pinnatifida ......................................................... 69
6.2.1 Introduction .............................................................................................................................. 69
6.2.2 Materials and methods .............................................................................................................. 69
6.2.3 Results and discussion .............................................................................................................. 69
7 CONCLUSIONS ........................................................................................................ 71
7.1 Summary of main findings ................................................................................... 71
7.2 Caveats ................................................................................................................. 73
7.3 Future research directions .................................................................................... 75
REFERENCES .............................................................................................................. 77
APPENDICES ............................................................................................................... 84
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9.1 L-fucose standard curve and linear regression ..................................................... 84
9.2 Sulphate standard curve and linear regression ..................................................... 85
9.3 Uronic acid standard curve and linear regression ................................................ 86
9.4 Protein standard curve and linear regression ....................................................... 87
9.5 Monthly variations in protein content of fucoidan ............................................... 88

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List of Figures

Figure 1 Structure of U. pinnatifida. ................................................................................ 5
Figure 2 The life cycle of U. pinnatifida .......................................................................... 6
Figure 3 Distribution of U. pinnatifida in New Zealand, and year it was first
sighted ...................................................................................................................... 7
Figure 4 Mark Allsopp, Wakatu Research Development Manager looks at an U.
pinnatifida-infested mussel line, Marlborough Sounds. .......................................... 8
Figure 5 Number of published articles on fucoidan-related work from 1980-2010. ..... 14
Figure 6 Pankter model of fucoidan ............................................................................... 15
Figure 7 Breast cancer rates across the world in 2008 ................................................... 22
Figure 8 The blade (left) and sporophyll (right) prior to drying. ................................... 27
Figure 9 Fucoidan content of U. pinnatifida with three extraction techniques. ............. 33
Figure 10 The colour of fucoidan extracted using CaCl extraction (A), water
2
extraction (B), and acid extraction (C) .................................................................. 34
Figure 11 Mussel farming sites in the Marlborough Sounds, New Zealand. ................. 40
Figure 12 Monthly variation in the percentage yield of crude fucoidan from U.
pinnatifida sporophyll (A) and blade (B) (106/327 = farm number) .................... 43
Figure 13 Monthly variations in the sulphate, uronic acid and fucose content in
fucoidan (106/327/353 = farm number). ............................................................... 45
Figure 14 Ion-exchange chromatography apparatus. ..................................................... 53
Figure 15 Calibration of gel permeation chromatography. ............................................ 56
Figure 16 Chromatogram of fucoidan on a DEAE-Sephadex A-25 column. ................ 57
Figure 17 Calibration curve for determination of the molecular weight of
fucoidan by Sephadex G-200................................................................................. 58
Figure 18 Scavenging effects on DPPH radical by crude fucoidan (F0), different
fucoidan fractions (FI, F2 and F3), commercial fucoidan (Sigma) and
Vitamin C............................................................................................................... 60
Figure 19 Total antioxidant capacity of crude fucoidan (F0), different fucoidan
fractions (FI, F2 and F3), and commercial fucoidan (Sigma) with the
CUPRAC reagent................................................................................................... 61
Figure 20 Commercial U. pinnatifida ‘Chung Jung Won’ Seaweed. ............................ 67
Figure 21 Comparison between U. pinnatifida which have been extracted using
CaCl for the first time (new) and U. pinnatifida which have been re-
2
extracted for the second time (old). ....................................................................... 70
Figure 22 Standard curve for the cysteine-sulphuric acid assay for the
determination of L-fucose concentration ............................................................... 84
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Figure 23 Standard curve for the BaCl -gelatin assay for the determination of
2
sulphate concentration expressed in K SO equivalents ....................................... 85
2 4
Figure 24 Standard curve for the carbazole assay for the determination of uronic
acid concentration expressed in glucuronic acid equivalents ................................ 86
Figure 25 Standard curve for the Bradford assay for the determination of protein
concentration expressed in bovine albumin equivalents ....................................... 87
Figure 26 Monthly variations in the protein content in fucoidan from the blade
(A) and sporophyll (B)........................................................................................... 88

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List of Tables

Table 1 Comparison of the previous and current policy for U. pinnatifida. .................. 11
Table 2 Yield and chemical composition of fucoidan extracted from different
species of seaweed. ................................................................................................ 19
Table 3 Composition and yield of crude fucoidan fractions from S. swartzii ................ 20
Table 4 Crude fucoidan fractions obtained from U. pinnatifida. ................................... 20
Table 5 Results (in percent of dry weight) from the chemical analysis of fucoidan
derived from three extraction methods. ................................................................. 35
Table 6 The month and farm where U. pinnatifida was harvested and extracted
for fucoidan ............................................................................................................ 41
Table 7 Chemical composition (% dry weight) of crude fucoidan (F0), its
fractions and commercial fucoidan (Sigma) .......................................................... 59
Table 8 Fucoidan in commercial seaweed. .................................................................... 68

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