Total Phenolic Content and Antioxidant Capacity of a selection of South African indigenous FRUITS.

145 Pages·2016·2.22 MB·English

TOTAL PHENOLIC CONTENT AND ANTIOXIDANT CAPACITY OF A
SELECTION OF SOUTH AFRICAN INDIGENOUS FRUITS

Daniela Amalia Kucich
Student No: 191031569

Dissertation submitted in partial fulfilment of the requirements for the degree
Master of Technology: Chemistry
in the Faculty of Applied Sciences
at the Cape Peninsula University of Technology

Supervisor: Merrill Wicht

Cape Town
May 2015

CPUT copyright information
The dissertation/thesis may not be published either in part (in scholarly, scientific or technical journals),
or as a whole (as a monograph), unless permission has been obtained from the University
Declaration

I, Daniela Amalia Kucich, declare that the contents of this dissertation/thesis represent my own
unaided work, and that the dissertation/thesis has not previously been submitted for academic
examination towards any qualification. Furthermore, it represents my own opinions and not
necessarily those of the Cape Peninsula University of Technology.

Signed Date

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Abstract

It has recently been confirmed that people consuming 7+ portions of fruit and vegetables daily
have a lower risk of mortality from any cause. With a fifth of the population of South Africa falling
below the poverty line, it has been found that rural adults have a very low daily intake of fruit and
vegetables; at the same time rural children are consuming a primarily maize-based diet. This low
dietary diversity translates into a higher level of infectious diseases in children younger than five
years. Interventions at national level included promoting the growing of underexploited
traditional indigenous vegetables and fruits in home gardens, in the hope that rural households
would help themselves in diversifying their cereal-based diet, while using crops they are
accustomed to in their environment.
Ten indigenous South African fruits found in the Western Cape were evaluated for their potential
to make a positive contribution to the diet of rural communities and were compared with
Blueberry and Cranberry, the North American ‘gold standards’. The following determinations
were carried out on 12 samples: Total Phenolic Content, Total Flavanols and Total Monomeric
Anthocyanins were analysed using the Folin-Ciocalteu, Mazza and pH Differential methods.
Total Antioxidant Capacity was assessed using the Trolox Equivalent Antioxidant Capacity
(TEAC), DPPH and Molybdenum Reduction assays. The Oxygen Radical Absorbance Capacity
(ORAC ) was also determined. Iron Chelating Activity, one of the methods recommended to
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reflect other antioxidant mechanisms, was also investigated.
The fruits possessing the highest concentration of Total Phenolic Content (Mazza) were
Christmas berry, Bietou, Wild Olive and Wild Plum, at levels significantly higher than those of the
two control berries, Blueberry and Cranberry. The fruits yielding the highest results for the
TEAC assay were Wild Plum, Wild Olive, Tortoise berry, Christmas berry and Colpoon. The
fruits giving the highest results for the DPPH assay were Wild Plum, Colpoon, Wild Olive, and
Christmas berry. The fruits showing the highest results for the Molybdenum Reduction assay
were Wild Olive, Wild Plum, Christmas berry, and Tortoise berry. The fruits yielding the highest
results for the ORAC Total Antioxidant Capacity assay were Colpoon, Christmas berry, Wild
Olive, Crossberry, Wild Plum, Waterberry followed by Blueberry and Cranberry. The results
from the Iron Chelating Activity assay revealed a ranking of Christmas berry, Blueberry, followed
by Num-num.
On combining the results of eight assays, namely TPC (Mazza), TF, TA, TEAC, DPPH, TAC,
TPC (FCR), ICA to give an Antioxidant Potency Composite Index, the fruits with the highest
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rankings were (1) Wild Plum, (2) Wild Olive, (3) Colpoon, and (4) Christmas berry. By
comparison the northern hemisphere control berries ranked (5) Blueberry and (9) Cranberry.
These findings show that by introducing even small servings of indigenous fruits into the diet, an
important and inexpensive source of natural antioxidants could be accessed and the mean daily
ORAC intake could thereby be boosted significantly by about 4,000 µmol Trolox Equivalents to
bring the Total ORAC consumed to within optimum levels (6,000 µmol Trolox Equivalents and
above). These bioactive plant compounds have the potential to deliver immense benefits to
health to impoverished South African adults, as well as rural children, well beyond basic
nutrition.

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Acknowledgements

I wish to thank

 Dr Nicolette Louw for inspiring me with her love of all things botanical
 Prof Victor Hugo for facilitating matters as supervisor
 Mrs Lorna Marshall for untiringly assisting me with methodologies
 Mrs Merrill Wicht for assuming the role of supervisor on the retirement of Prof Hugo
 Prof Jeanine Marnewick for her invaluable insight and input from the literature
 Prof Wentzel Gelderblom for his guidance and overview when this project was initially
undertaken
 Ms Petra Snijman for the training offered through the MRC in the TEAC method
 Mr Jaco Kotze for the assays undertaken
 Mr Fanie Rautenbach for his assistance with the ORAC method
 CPUT for the seed funding received

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Dedication

For Roberto, my champion…

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Table of Contents
Declaration................................................................................................................................................................................... ii
Abstract ........................................................................................................................................................................................ iii
Acknowledgements .................................................................................................................................................................... v
Dedication ................................................................................................................................................................................... vi
Table of Contents ..................................................................................................................................................................... vii
Glossary ..................................................................................................................................................................................... xiii
CHAPTER 1 INTRODUCTION ............................................................................................................. 1
CHAPTER 2 SELECTED INDIGENOUS BOTANICAL SPECIES .................................................... 9
Syzygium cordatum ................................................................................................................................................................. 10
Osyris compressa ...................................................................................................................................................................... 11
Harpephyllum caffrum ........................................................................................................................................................... 12
Chironia baccifera .................................................................................................................................................................... 13
Carissa macrocarpa ................................................................................................................................................................ 15
Nylandtia spinosa ..................................................................................................................................................................... 16
Carpobrotus edulis ................................................................................................................................................................... 17
Chrysanthemoides monilifera .............................................................................................................................................. 18
Grewia occidentalis.................................................................................................................................................................. 19
Olea europaea subsp. africana ............................................................................................................................................ 20
CHAPTER 3 THE LINK BETWEEN DIET, ANTIOXIDANTS AND HEALTH .......................... 21
How did we arrive at the concept of ‘antioxidant status’? ........................................................................................ 21
The epidemiology puzzle ...................................................................................................................................................... 22
The Major Classes of Plant Phenolics ............................................................................................................................... 23
What the Epidemiology studies revealed ....................................................................................................................... 28

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CHAPTER 4 CHOOSING FROM A PLETHORA OF ANTIOXIDANT CAPACITY ASSAYS ... 35
The body’s defence system against ROS .......................................................................................................................... 38
Evaluation of natural antioxidants ................................................................................................................................... 39
Classification of antioxidants by reaction mechanism............................................................................................ 43
Classification of AOC assays by HAT or SET reaction mechanisms (10) ............................................................... 43
HAT-based methods: ........................................................................................................................................................... 44
SET-based methods: ............................................................................................................................................................ 45
Detailed discussion of the recommended AOC methods using HAT reaction mechanisms ......................... 46
ORAC: Oxygen Radical Absorbance Capacity .............................................................................................................. 46
Detailed discussion of recommended AOC methods using both HAT and SET reaction mechanisms ..... 49
TEAC or other ABTS⋅+ assays (26): ..................................................................................................................................... 49
DPPH (9): .................................................................................................................................................................................. 51
Total Phenolic Content - Folin Ciocalteu Reagent: .................................................................................................... 53
Iron Chelating Activity ICA: .............................................................................................................................................. 54
Validation issues: ..................................................................................................................................................................... 56
Standardization of AOC methods: ...................................................................................................................................... 56
Summation: Generating an ’Antioxidant Profile’ for natural materials: ............................................................. 56

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CHAPTER 5 RESEARCH METHODOLOGY ................................................................................... 58
Sampling of berries: ................................................................................................................................................................ 58
Extraction of berries: ............................................................................................................................................................. 58
Chemicals and apparatus: .................................................................................................................................................... 58
Methods ....................................................................................................................................................................................... 59
1.Total Phenolic Content (Folin Ciocalteu Reagent): ............................................................................................... 59
2.Total Phenolic Contents (Mazza method): ............................................................................................................... 60
3.Total Flavonols: ................................................................................................................................................................ 60
4.Total Monomeric Anthocyanins: ................................................................................................................................. 60
5. +) Assay: ...................................................................................................................................................... 61
6.DPPH Assay: ....................................................................................................................................................................... 62
7.Molybdenum Reduction assay: .................................................................................................................................... 63
8.ORAC method: .................................................................................................................................................................... 64
9.Iron Chelating Activity ICA: ........................................................................................................................................... 65
CHAPTER 6…RESULTS AND DISCUSSION ..................................................................................... 67
Results: Phenolic Profiles .................................................................................................................................................. 68
Results: Antioxidant capacity as TEAC, DPPH & Molybdenum Reduction assays ........................................... 74
Results: Antioxidant Capacity as ORAC and TPC (FCR) ........................................................................................... 79
Results: Iron Chelating Activity ....................................................................................................................................... 83
Statistical analysis .............................................................................................................................................................. 84
Discussion .............................................................................................................................................................................. 88
CHAPTER 7…CONCLUSION ............................................................................................................... 95

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APPENDIX ............................................................................................................................................. 111
Examples of Calibration Curves and Calculations .................................................................................................... 111
1.Total Phenolic Content (Mazza) ............................................................................................................................... 111
2.Total Flavonols ............................................................................................................................................................... 114
3.Total Monomeric Anthocyanins ............................................................................................................................... 116
4.TEAC ................................................................................................................................................................................... 117
5.DPPH .................................................................................................................................................................................. 120
6.Molybdenum Reduction............................................................................................................................................... 123
7.ORAC .................................................................................................................................................................................. 126
8.Total Phenolic Content (FCR) .................................................................................................................................... 128
9.Iron Chelating Activity ................................................................................................................................................. 130

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