THESIS MOHD SHUKRI MAT ALI 2008 - Glasgow Theses Service

200 Pages·2008·14.97 MB·English

Analysis of Phenolics and Other
Phytochemicals in Selected Malaysian
Traditional Vegetables and Their Activities
In Vitro

Mohd Shukri Mat Ali

BSc (Hons), MSc. (Biochemistry)

A thesis submitted to the Faculty of Biomedical and Life Sciences,
University of Glasgow, for the degree of
Doctor of Philosophy (PhD)
ABSTRACT

A fruit and vegetable-rich diet has been associated with decreased risk of developing
chronic diseases such as cardiovascular disease and cancer in humans. These protective
effects have been attributed in part, to the presence of phytochemicals in fruit and
vegetables, in particular flavonoids and phenolic compounds. Some plants have been used
in traditional medicine for healing, ritual ceremonies and as health tonics or food
supplements. Recent interest in the health-promoting properties of Malaysian traditional
vegetables has been based on claims about their uses in health and medicine. However,
scientific information to support these claims is largely unexplored. The overall objectives of
the present study were to investigate, determine and quantify the phytochemicals,
particularly phenolic compounds, in the seven samples from five species of selected
Malaysian traditional vegetables (Anacardium occidentale, Centella asiatica, Colubrina
asiatica, Pluchea indica and Premna cordifolia) and to evaluate their activities in vitro,
including antioxidant and antibacterial activities of extracts of these plants and individual
phytochemicals. In the first section of this project, discussed in Chapter 3, Malaysian
traditional vegetable extracts were screened for phenolic compounds using several
complimentary techniques, namely high performance liquid chromatography (HPLC) and
HPLC-tandem mass spectrometry and the total phenolic content determined using the
Folin-Ciocalteu assay. Flavonol glycosides were predominant in most of the species,
particularly A. occidentale with levels ranging from 6434 to 12420 µg/g fresh weight.
Chlorogenic acids were the main components identified and quantified in C. asiatica and P.
indica. The total phenolic content of the vegetables were between 100 ± 7.8 and 415 ± 20
mg/ kg gallic acid equivalent (GAE) in batch 1 but lower in batch 2 ranging from 62 ± 2.5 to
386 ± 41 mg/ kg GAE. The total phenolic content of plant extracts was positively correlated
with total antioxidant capacity, determined by 2, 2’-azinobis-3-ethylbenzothiazoline-6-
sulfonic acid (ABTS) and ferric reducing antioxidant potential (FRAP) assays.

A. occidentale exhibited the highest total phenolic content and total antioxidant
activity, whereas Colubrina asiatica, which had the lowest total phenolic content, also had
low antioxidant activity in vitro. Phenolic content and antioxidant activity were significantly
(p<0.05) influenced by environmental factors, as in this study, plant materials in batch 1
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which was harvested in rainy season, had a higher total phenolic and antioxidant content
than batch 2, which was harvested in the dry season.

Based on the hypothesis that other components in addition to phenolics also
contributed to the total antioxidant activities in the plants, the next objective, which was
presented in Chapter 4, was to investigate the occurrence of phytochemicals such as
triterpenes, carotenoids, α-tocopherol and vitamin C. The level of total triterpenes,
biomarkers of C. asiatica was not significantly different between batches. The main
component was madecassoside with 91 ± 4.8 µg/g fresh weight in batch 1 and 77 ± 3.4
µg/g fresh weight in batch 2. The level of carotenoids and vitamin C were low compared to
previous reports. This was almost certainly due to dried samples being used in the present
study, as some of the compounds would have broken down during drying process. This
would have particularly affected the levels of vitamin C, which contributed only 0.9 to 5.5%
to the total antioxidant activity of the plants under study.

Total antioxidant activities of plant essential oils were determined using 1, 1-
diphenyl-2-picrylhydrazyl (DPPH) and the result was in agreement to the total antioxidant
activities of plant extracts, which A. occidentale having the highest amount. The highest
antioxidant activity exhibited by A. occidentale oil was attributed to the presence of high
amounts of γ-terpinene (28%) and terpinen-4-ol (4.2%), both of which were shown to have
strong radical scavenging activity.

The high phenolic content, antioxidant activity and occurrence of volatile
components exhibited by A. occidentale has led to the final objective of this study, which is
presented in Chapter 5. This was to screen for antimicrobial activities of A. occidentale
extracts and essential oil against selection of Gram-positive (Enterococcus faecalis,
Staphylococcus aureus, Meticillin-resistance Staphylococcus aureus (MRSA), coagulase
negative Staphylococci (CoNS) and Lactobacillus acidophilus), Gram-negative bacteria
(Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and fungi
(Candida albican) using disc diffusion and minimum inhibitory concentration (MIC) methods.
Investigation of the modes of action was determined using growth inhibition curve, scanning
(SEM) and transmission (TEM) electron microscopy. A. occidentale was shown to have
promising effects at 25 mg/ml with regard to inhibiting the growth of Gram-positive bacteria
including MRSA. The essential oil and its major component, γ-terpinene at only 2.5% (v/v)
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inhibited the growth of all Gram-positive and Gram-negative bacteria. None of the A.
occidentale extracts or oil exhibited antibacterial activities against Lactobacillus acidophilus,
an important strain of bacteria found in the human gut. This indicates selective effects of A.
occidentale.

A. occidentale extract and oil inhibited the growth of S. aureus cells within a 2-hour
incubation observed in time-kill experiments. SEM and TEM examination revealed that the
oil and its component, γ-terpinene, inhibited the bacteria through bacteriostatic and
bactericidal effects which damaged the bacterial cell wall. Testing the oil and γ-terpinene
against epidemic-MRSA (EMRSA) biofilms indicated an anti-adhesive effect, which
disrupted the bacterial colonies in the biofilms to produce more extracellular
polysaccharides (EPS). The effects of A. occidentale oil were comparable with tea tree oil, a
widely used topical antiseptic.

All the Malaysian traditional vegetables under study are claimed to have medicinal
properties and health effects. The results in the present study have provided some
information on phytochemical and nutritional properties of Malaysian traditional vegetables,
and as a consequence provide a sound scientific base for promoting their consumption
particularly in Malaysia.

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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iv
ACKNOWLEDGEMENTS xi
AUTHOR’S DECLARATION xii
ABBREVIATIONS xiii
LIST OF TABLES xv
LIST OF FIGURES xvii
AIMS OF STUDY xx

CHAPTER 1 : INTRODUCTION
1.1 Medicinal plants, herbs, traditional vegetables and exotic vegetables 1
1.2 Malaysian traditional vegetables 3
1.2.1 Anacardium occidentale L. (Anacardiaceae) 7
1.2.2 Centella asiatica (L.) Urban (Umbelliferae) 8
1.2.3 Colubrina asiatica (L.) Brongn (Rhamnaceae) 9
1.2.4 Pluchea indica (L.) Lees. (Compositae) 10
1.2.5 Premna cordifolia Roxb. (Verbanaceae) 11
1.3 Fruit and vegetables and health effects 12
1.4 Phytochemicals in plants 14
1.4.1 Phenolic compounds - Flavonoids 14
1.4.1.1 Flavonols 16
1.4.1.2 Flavones 17
1.4.1.3 Isoflavones 21
1.4.1.4 Flavanones 21
1.4.1.5 Flavan-3-ols 21
1.4.1.6 Anthocyanidins 22
1.4.2 Chlorogenic acids 23
1.4.3 Vitamins 24
1.4.3.1 Carotenoids as pro-vitamin A 24
1.4.3.2 Vitamin C 26
1.4.3.3 Vitamin E 27
1.4.4 Alkaloids 27
1.4.5 Plant essential oils and volatile compounds 28
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1.5 Phytochemicals and health effects 29
1.5.1 Flavonoids, dietary intake, bioavailability, absortion and health 30
effects
1.5.2 Vitamins and health effects 33
1.6 Environmental effects on the level of phytochemicals 33
1.7 Biological activities 34
1.7.1 Antioxidants 35
1.7.2 Anticancer 37
1.8 Future drugs from plants 38

CHAPTER 2 : MATERIALS AND METHODS
2.1 Planting materials 39
2.2 Chemicals and reagents 39
2.3 Extraction of plant materials 42
2.3.1 General extraction for phytochemical analysis 42
2.3.2 Extraction for phytochemical analysis and antimicrobial screening 42
2.2.3 Extraction for GC-MS analysis 42
2.4 Determination of total phenolic content 43
2.5 Extraction and analysis of carotenoids and α-tocopherol 43
2.6 Extraction and analysis of ascorbic acid 44
2.7 HPLC-PDA-MS2 analysis of selected Malaysian traditional vegetables 45
2.8 HPLC and HPLC-PDA-MS2 analysis of triterpenes in Centella asiatica 46
2.9 GC-MS analysis of essential oils of selected Malaysian traditional 46
vegetables
2.10 2,2’-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) 46
decolourisation assay
2.11 Ferric reducing antioxidant potential (FRAP) assay 47
2.12 Measurement of radical-scavenging activity by 1,1-diphenyl-2- 48
picrylhydrazyl (DPPH) colourimetry assay for essential oils
2.13 Statistical analysis 49

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CHAPTER 3 : ANALYSIS OF PHENOLICS IN SELECTED MALAYSIAN TRADITIONAL
VEGETABLES
3.1 Introduction 50
3.2 HPLC-tandem mass spectrometry analysis of phenolic compounds in 52
selected Malaysia traditional vegetables
3.2.1 HPLC-PDA-MS2 analysis of phenolics in Anacardium occidentale 52
3.2.2 HPLC-PDA-MS2 analysis of phenolics in Centella asiatica 56
3.2.3 HPLC-PDA-MS2 analysis of phenolics in Colubrina asiatica 60
3.2.4 HPLC-PDA-MS2 analysis of phenolics in Pluchea indica 64
3.2.5 HPLC-PDA-MS2 analysis of phenolics in Premna cordifolia 67
3.3 Levels of phenolic compounds in Malaysian traditional vegetables: 70
comparison of two batches
3.4 Total antioxidant activities of Malaysian traditional vegetables 76
3.4.1 Correlations between total phenol and total antioxidant activities 76
3.5 Discussion 79
3.6 Conclusion 84

CHAPTER 4 : ANALYSIS OF OTHER PHYTOCHEMICALS IN SELECTED MALAYSIAN
TRADITIONAL VEGETABLES
4.1 Introduction 86
4.2 HPLC and HPLC-tandem mass spectrometry analysis of other 88
phytochemicals in selected Malaysian traditional vegetables
4.2.1 Qualitative and quantitative analysis of triterpenes in Centella 88
asiatica
4.2.2 Level of carotenoids and α-tocopherol 91
4.2.3 Level of vitamin C 97
4.3 GC-tandem mass spectrometry analysis of the volatile compounds in 98
selected Malaysian traditional vegetables
4.4 Measurement of antioxidant activities of vitamin C and volatile 109
compounds
4.4.1 Measurement of vitamin C antioxidant using FRAP-ascorbic acid 109
assay
4.4.2 Measurement of antioxidant activities of volatile compounds 110
using DPPH assay

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4.5 Discussion 112
4.5.1 Analysis of other phytochemicals in selected Malaysian traditional 112
vegetables
4.5.2 Analysis of volatile components 115
4.5.3 Analytical methodologies for quantitative analysis of Malaysian 117
vegetables
4.5.4 Influence of environmental factors and varieties on the level of 119
phytochemicals in Malaysian vegetables
4.5.5 Total antioxidant activities of vitamin C and volatile compounds 120
4.6 Conclusion 122

CHAPTER 5 : ANALYSIS OF OTHER PHYTOCHEMICALS IN SELECTED MALAYSIAN
TRADITIONAL VEGETABLES
5.1 INTRODUCTION 123
5.1.1 Bacteria 123
5.1.2 Bacterial infections 124
5.1.2.1 Community-acquired infections 124
5.1.2.2 Nosoconial infections 125
5.1.3 Staphylococcus species 127
5.1.3.1 Meticillin-resistant Staphylococcus aureus 128
5.1.3.2 Coagulase-negative staphylococci 129
5.1.4 Enterococcus species 130
5.1.5 Gram-negative bacilli 130
5.1.6 Normal flora 131
5.1.7 Biofilms 132
5.1.8 Biofilms, medical devices, MRSA infection and antibiotic 133
challenges
5.1.9 Novel compounds from plant as antimicrobial agents 134
5.1.9.1 Antimicrobial activity of phytochemicals 135
5.1.9.2 Antimicrobial activity of plant volatiles 138
5.1.10 Evaluation of antimicrobial activity 139
5.1.10.1 Colourimetric assay to measure biomass and biofilms 139
5.1.10.2 XTT assay to measure biofilm viability 140
5.1.11 Modes of action and mechanism of inhibition of antimicrobial 140
agents
5.1.12 Aims of this chapter 141

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5.2 MATERIALS AND METHODS 143
5.2.1 PART 1: THE EFFECTS OF MALAYSIAN TRADITIONAL 143
VEGETABLES EXTRACTS AND OILS AGAINST PLANKTONIC
BACTERIA
5.2.1.1 Samples 143
5.2.1.2 Reagents and bacteria 143
5.2.1.3 Antimicrobial susceptibility test by disc diffusion 143
method
5.2.1.4 MIC 145
5.2.1.5 Bacterial growth measurement 145
5.2.1.6 Scanning electron microscope (SEM) and transmission 146
electron microscope (TEM) analysis for the observation
of bacteria structures and bacteria cell walls
5.2.2 PART 2: THE EFFECTS ANACARDIUM OCCIDENTALE 147
EXTRACTS AND OIL AGAINST EMRSA BIOFILMS
5.2.2.1 Extracts and oil of A. occidentale and Melaleuca 147
alternifolia
5.2.2.2 Isolates used and growth condition 147
5.2.2.3 Preparation of XTT (metabolic substrate) and 147
menadione (electron coupler)
5.2.2.3.1 XTT optimisation 148
5.2.2.3.2 Determination of optimal XTT incubation 149
time
5.2.2.4 Effects of plant extracts on MRSA biofilms 149
5.2.2.5 Quantification of the biofilm biomass 150
5.2.2.6 Total viable cell counts 150
5.2.2.6.1 Total viable counts of planktonic cell 150
5.2.2.6.2 Total viable counts of biofilms 150
5.2.2.7 Scanning electron microscopy analysis of biofilms with 151
and without treatment
5.3 RESULTS
5.3.1 PART 1: THE EFFECTS OF MALAYSIAN TRADITIONAL 152
VEGETABLES EXTRACTS AND OILS AGAINST PLANKTONIC
BACTERIA
5.3.1.1 Disc diffusion method to assess antimicrobial property 152
of crude extracts of Malaysian traditional vegetables on
selected bacterial strains
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5.3.1.2 Disc diffusion method to assess antimicrobial activity of 152
crude extracts of A. occidentale on MRSA and
coagulase-negative staphylococci
5.3.1.3 Determination of MIC to assess antimicrobial activity of 154
A. occidentale extracts against Gram-positive bacteria
5.3.1.4 Disc diffusion method to assess antimicrobial property 154
of essential oils of Malaysian traditional vegetables on
selected bacterial strains
5.3.1.5 Determination of MIC to assess antimicrobial activity of 158
A. occidentale essential oil
5.3.1.6 Assessment of the antimicrobial property of essential oil 158
of A. occidentale and its components on selected bacterial
strains
5.3.1.7 The effects of selected A. occidentale crude extracts 161
and essential oil on Lactobacillus spp.
5.3.1.8 The effects of A. occidentale crude extracts and 161
essential oil on fungi (Candida albicans)
5.3.1.9 Time-kill experiments: The effect of A. occidentale 164
crude extracts and oil on S. aureus ATCC 29213
growth against exposure time
5.3.1.10 Physical valuation of the effect of plant extracts and 167
oils on S. aureus ATCC 29213 by SEM and TEM
5.3.2 PART 2: THE EFFECTS ANACARDIUM OCCIDENTALE 170
ESSENTIAL OIL AGAINST EMRSA BIOFILMS
5.3.2.1 Optimisation of XTT assay 170
5.3.2.1.1 Determination of optimal menadione 170
concentration in planktonic cells
5.3.2.1.2 Determination of optimal XTT-menadione 170
incubation time in planktonic cells
5.3.2.1.3 The effect of planktonic cell density on the 173
XTT-absorbance
5.3.2.2 The effects of essential oils of A. occidentale and M. 173
alternifolia on the viability of EMRSA biofilms
5.3.2.3 The effects of essential oils of A. occidentale and M. 173
alternifolia on the biomass of EMRSA biofilms
5.3.2.4 Physical valuation of the effect of the essential oils on 177
EMRSA biofilms by SEM

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