AlGamdi, Noura Ahmad (2013) Characterisation of polyphenolic compounds in herbal tea and ...

206 Pages·2013·2.93 MB·English

AlGamdi, Noura Ahmad (2013) Characterisation of polyphenolic
compounds in herbal tea and berries: in vivo and in vitro studies on the
bioavailability of anthocyanins. PhD thesis.

http://theses.gla.ac.uk/4768/

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Characterisation of polyphenolic compounds
in herbal tea and berries: in vivo and in vitro
studies on the bioavailability of anthocyanins

Noura Ahmad AlGamdi
BSc (Hons), MSc (Botany)

April, 2013

A thesis submitted to the School of Medicine, College of Medical,
Veterinary and Life science, University of Glasgow for the degree
of Doctor of Philosophy (PhD)
This thesis is dedicated to my family:

A special feeling of gratitude to my loving parents, whose endless love, care,
encouragement fills me with joy, strength and tenacity each day of my life. I’m
so blessed to have you both.

To my true love, my husband, best friend and soul mate. Your love, support
constant patience and sacrificing inspired me to complete this work as you
made our future look brighter.

To my brothers and sisters, who have prayed for me and were by my side
throughout all the process. I will always appreciate all what they have done.

At the end, I also dedicate this dissertation to my coming little angel, my baby,
where another journey begins.
iii
Abstract

Flavonoids and polyphenolic compounds are widely distributed in commonly
consumed foods, beverages and herbs. Their consumption has been associated
with improving the health and reducing the risk of several chronic diseases. In
Saudi Arabia, recent interest in the health-promoting properties of traditional
herbs has been increased. To-date researchers have focused on testing these
therapeutic properties. However, information about their chemical profile is
largely unexplored. Thus, the beginning of this project aimed to use HPLC–PDA–
MS2 to identify polyphenolic compounds in a medicinal herbal tea made from
seeds of Anastatica hirerochuntica. Twenty compounds comprising a series of
flavone C- and O-linked glycosides, phenolic acids, chlorogenic acids and
flavonols were identified or partially identified on the basis of co
chromatography with reference compounds and MS2 and MS3 fragmentation
patterns. The flavones were the principal components, occurring as luteolin,
apigenin and diosmetin conjugates. The antioxidant potential of individual
compounds in Anastatica was assessed using HPLC with an on-line ABTS•+
detection system. Under the experimental conditions, 14 compounds exhibited
antioxidant activity. The highest contribution to the antioxidant capacity of the
tea, 56%, came from 3,4-dihydroxybenzoic acid and caffeoyl- and
dicaffeoylquinic acids while 6-C-glucosides of luteolin and apigenin contributed
41%. These findings together with tests in the FRAP antioxidant assay and the
Folin–Ciocalteu total phenolics assay revealed that aqueous infusion of A.
hirerochuntica seed is rich in polyphenolic compounds.

Another perspective of investigating these polyphenolic compounds is not just
knowing their chemical constituents but also studying their bioavailability upon
ingestion. Therefore, blackberries and strawberries were chosen for intervention
studies with human subjects to examine the bioavailability of anthocyanins, a
group of flavonoids, due to their importance as natural antioxidants that have
been linked with beneficial effects on health to humans. In the first in vivo
study, plasma and urine were collected from eight healthy subjects after
ingestion of 180 g of blackberries and analysed by HPLC-PDA-MS2. Cyanidin-3-O-
glucoside was metabolised principally to peonidin-O-glucuronide. Cyanidin-3-O-
iv
glucoside and its metabolites appeared rapidly in the plasma in low
concentrations ranging from 5 to 20 nmol/L with low urinary excretion of ~0.08%
of the total intake. In the second in vivo study, both blackberry and strawberry
were used to examine the small intestinal absorption and metabolism of both
cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside by feeding five ileostomy
volunteers with 180 g of berries. Samples of urine and ileal fluid were collected
over a 0-24 h period after ingestion and analysed by HPLC-PDA-MS2
Unmetabolized anthocyanins of blackberries and strawberries were recovered in
ileal fluids at a level of 5.1% and 13.6%. This indicates either low level of
absorption or lack of stability at the non-acid pH in the small intestine. The
overall urinary excretion of anthocyanins was 0.06% and 0.87% of intake of
blackberries and strawberries respectively. The urine data shows that both
cyanidin and pelargonidin glucosides were metabolised primarily to peonidin-O-
glucuronide and pelargonidin-O-glucuronide respectively. Although low
recoveries of the two compounds are reported in this investigation in both ileal
fluid and urine, pelargonidin-3-O-glucoside showed a slightly higher recovery
compared to cyanidin-3-O-glucoside. The data from both in vivo studies reflect
the influence of the aglycone structure on the bioavailability of these
anthocyanins.

The stability of anthocyanins in the gastrointestinal tract was also assessed using
in vitro models (Chapter 5). The results indicated that at neutral pH the salivary
enzymes and oral microbiota had no impact on cyanidin-3-O-glucoside but
degraded pelargonidin-3-O-glucoside by ~20%. With simulated and human gastric
juice, both anthocyanins were unstable and the breakdown continued
dramatically under pancreatic digestion where a 5.1% recovery was obtained.
Their low recovery levels were comparable to those obtained in vivo with ileal
fluid. These findings imply that the chemical structure of both anthocyanins
influence their stability similarly. However, the data provide no explanation
about the higher bioavailability of pelargonidin-3-O-glucoside compared to
cyanidin-3-O-glucoside in vivo. This presumably is a consequence of the more
ready access of pelargonidin-3-O-glucoside to the enterocytes of the small
intestine.
v
In conclusion, the work here has shown the diversity of compounds with
potential health benefits that are present in a natural product from Saudi
Arabia. Experiments were carried out, using local products from the UK, to
investigate factors that would affect their potential to provide health benefits.
The in vivo and in vitro experiments show the complexity of investigations of
potential bioactive compounds in human nutritional research. Polyphenols which
differ by only a single hydroxyl group show very different absorption,
metabolism and stability properties.
vi
Table of Contents

Abstract iii
List of tables x
List of figures xiii
Abbreviations xv
Acknowledgment xvii
Author’s declaration xviii
1 Introduction 1
1.1 Phytochemicals in plants 1
1.1.1 Phenolic and polyphenolic compounds 2
1.1.1.1 Flavonoids 2
1.1.1.2 Flavonols 3
1.1.1.3 Flavones 4
1.1.1.4 Flavan-3-ols 5
1.1.1.5 Flavanones 6
1.1.1.6 Isoflavones 6
1.1.1.7 Anthocyanidins 7
1.1.2 Non-flavonoids 8
1.1.2.1 Phenolic acids 8
1.1.2.2 Hydroxycinnamates 8
1.1.2.3 Stilbenes 8
1.2 Dietary flavonoids and health 9
1.3 Medicinal herbal plants 11
1.4 Anthocyanins as natural pigments in plants: structure and occurrence 16
1.5 Stability of anthocyanins 17
1.5.1 Glycosylation and acylation 17
1.5.2 pH effect 19
1.5.3 Other factors affecting anthocyanins stability 21
1.6 Anthocyanins in berries 23
1.7 Bioavailability and metabolism of anthocyanins 27
1.7.1 Bioavailability definition 27
1.7.2 Bioavailability studies of anthocyanins 30
1.7.2.1 Animal studies 30
1.7.2.2 Human studies 31
vii
1.7.3 Factors affecting anthocyanin bioavailability 32
1.7.4 Anthocyanins absorption and metabolism 35
1.7.4.1 Absorption of anthocyanins 36
1.7.4.2 Metabolism of anthocyanins 38
1.8 Aims and objectives of the study 38
1.8.1 Background 38
1.8.2 Aims 41
1.8.3 Objectives 41
2. Profiling of flavonoids and phenolic compounds in Anastatica
hirerochuntica seeds by HPLC-PDA-MS2 43
2.1 Introduction 43
2.2 Materials and methods 47
2.2.1 Chemicals and reagents 47
2.2.2 Preparation of Anastatica hirerochuntica extract 47
2.2.3 Colorimetric assays 48
2.2.3.1 Measurement of antioxidant activity using ferric reducing
antioxidant potential (FRAP) assay 48
2.2.3.2 Determination of total phenol content 48
2.2.4 HPLC with diode array detection and tandem mass spectrometry 49
2.2.4.1 Analysis of phenolics compounds for Anastatica hirerochuntica
extract 49
2.2.4.2 Trolox equivalent antioxidant capacity (TEAC) assay and on-
line ABTS•+ assay systems 50
2.2.5 Data analysis 51
2.3 Results and discussion 52
2.3.1 Qualitative analysis of A. hirerochuntica extract 52
2.3.2 Quantitative analysis of A. hirerochuntica extract 59
2.3.3 On-line ABTS•+ analysis of antioxidants 60
2.3.4 Antioxidant capacity and total phenol content 64
2.4 Conclusion 64
3. Bioavailability of anthocyanins following consumption of blackberries by
healthy subjects 66
3.1 Introduction 66
3.2 Materials and methods 68
viii
3.2.1 Chemicals and reagents 68
3.2.2 Study design 68
3.2.3 Preparation of blackberry extract 69
3.2.4 Analysis of anthocyanins in biological fluids 70
3.2.4.1 Extraction of anthocyanins from plasma 70
3.2.4.2 Extraction of anthocyanins from urine 70
3.2.5 Pharmacokinetic analysis of anthocyanins in plasma 71
3.2.6 HPLC with diode array detection and tandem mass spectrometry 71
3.2.7 Data analysis 72
3.3 Results 72
3.3.1 Analysis of blackberries 72
3.3.2 Identification and quantification of anthocyanins in plasma 77
3.3.3 Identification and quantification of anthocyanins in urine 81
3.4 Discussion 86
3.5 Conclusion 95
4. Bioavailability of anthocyanins following consumption of blackberries and
strawberries by ileostomy subjects 97
4.1 Introduction 97
4.2 Materials and methods 98
4.2.1 Chemicals and reagents 98
4.2.2 Preparation of blackberry and strawberry extract 99
4.2.3 Study design 99
4.2.4 Analysis of anthocyanins in biological fluids 99
4.2.4.1 Extraction of anthocyanins from ileal fluids 99
4.2.4.2 Extraction of anthocyanins from urine 100
4.2.5 HPLC with diode array detection and tandem mass spectrometry 100
4.2.6 Data analysis 101
4.3 Results 101
4.3.1 Blackberry study 101
4.3.1.1 Analysis of blackberries 101
4.3.1.2 Identification and quantification of blackberry anthocyanins in
ileal fluid 102
4.3.1.3 Identification and quantification of blackberry anthocyanins in
urine 107
4.3.1.4 Urinary excretion in healthy and ileostomist groups 110
ix
4.3.2 Strawberries study 111
4.3.2.1 Analysis of strawberries 111
4.3.2.2 Identification and quantification of anthocyanins in ileal fluid
after ingestion of strawberries 116
4.3.2.3 Identification and quantification of anthocyanins in urine after
ingestion of strawberries 121
4.3.2.4 Breakdown of anthocyanins in urine 125
4.4 Discussion 126
4.5 Conclusion 133
5. Gastrointestinal stability of cyanidin-3-O-glucoside and pelargonidin-3-O-
glucoside in vitro. 136
5.1 Introduction 136
5.2 Materials and Methods 138
5.2.1 Chemicals and reagents 138
5.2.1 Preparation of blackberry and strawberry extracts 138
5.2.2 In vitro digestion model 138
5.2.3 Incubation condition 139
5.2.3.1 Saliva incubation 139
5.2.3.2 Simulated gastric and pancreatic juices incubation 139
5.2.4 Human gastric juice incubation 140
5.2.5 HPLC with diode array detection and tandem mass spectrometry 140
5.2.6 Data analysis 141
5.3 Results 141
5.3.1 Saliva incubation with cyanidin-3-O-glucoside and pelargonidin-3-O-
glucoside 141
5.3.2 Human gastric and simulated gastric juice incubation 143
5.3.3 Simulated pancreatic juices incubation 146
5.4 Discussion 149
5.5 Conclusion 154
6 General discussion 156
7 Conclusion and recommendations 162
Appendices 165
List of references 166