METABOLIC CHANGES IN DIET-INDUCED OBESITY AMONG RATS TREATED WITH ETHANOL EXTRACT OF Alternanthera sessilis RED AZIZAH BINTI OTHMAN FPSK(p) 2016 16
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METABOLIC CHANGES IN DIET-INDUCED OBESITY AMONG RATS TREATED WITH ETHANOL EXTRACT OF Alternanthera sessilis RED
AZIZAH BINTI OTHMAN
FPSK(p) 2016 16
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METABOLIC CHANGES IN DIET-INDUCED OBESITY AMONG RATS
TREATED WITH ETHANOL EXTRACT OF Alternanthera sessilis RED
By
AZIZAH BINTI OTHMAN
Thesis submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfillment of the Requirements for the Degree of Doctor of Philosophy
May 2016
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COPYRIGHT
All material contained within the thesis, including without limitation text, logos,
icons, photographs and all other artwork, is copyright material of Universiti Putra
Malaysia unless otherwise stated. Use may be made of any material contained within
the thesis for non-commercial purposes from the copyright holder. Commercial use
of material may only be made with the express, prior, written permission of
Universiti Putra Malaysia.
Copyright © Universiti Putra Malaysia
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment
of the requirement for the degree of Doctor of Philosophy
METABOLIC CHANGES IN DIET-INDUCED OBESITY AMONG RATS
TREATED WITH ETHANOL EXTRACT OF Alternanthera sessilis RED
By
AZIZAH BINTI OTHMAN
May 2016
Chairman : Professor Amin Ismail, PhD
Faculty : Medicine and Health Sciences
Alternanthera sessilis or also known by the name of ‗keremak‘ in Malaysia is a
perennial plant that belongs to the plant family of Amaranthaceae. Less research has
been conducted on local Alternanthera sessilis species due to its low commercial
value. This study was carried out to investigate the nutritional composition,
antioxidant capacities and phenolic compounds of Alternanthera sessilis red (ASR)
and Alternanthera sessilis green (ASG) in water and ethanolic extracts grown in
Malaysia. The ASR has been consumed traditionally as herbal drinks. However, very
little scientific data reported about the effect of this plant towards health. Thus, this
study was examined the effect of low (175 mg/kg BW) and high (350 mg/kg BW)
doses ASR ethanolic extracts supplementation on plasma lipid profile, leptin,
adiponectin, liver and kidney function test of diet-induced obesity rats. If an obese rat
treated with ASR ethanolic extract, then it will alter the urinary and serum
metabolites. Therefore a NMR spectroscopy in conjunction with multivariate
analysis was applied to examine metabolite changes and obesity biomarkers in urine
and serum of diet-induced obesity rats. The results showed that ASR contained
significantly (p < 0.05) higher proximate and mineral contents compared to ASG.
Ethanolic extracts of both ASR and ASG presented significantly greater antioxidant
capacity, than the water extracts at p < 0.05. Total phenolic content exhibited a
positive correlations with antioxidant capacities which could be one of the critical
contributors to the antioxidant activity. A total of eleven phenolic compound were
identified in ASR extract. The phenolic compound in ASR extract were gallic acid,
epigallocatechin, catechin, chlorogenic acid, 4-hydroxybenzoic acid, vanilic acid,
ferulic acid, ethyl gallate, myricetin, daidzein and apigenin. The low dose ASR
extract significantly improved plasma lipid profile and decreases leptin level after
eight weeks of daily therapy. While obese rats treated with high dose ASR extract
have significantly fewer visceral fat mass compared to other studied groups. Obesity
due to high fat diet led to an increase in urinary glucose, creatinine, serotonin and 3-
hydroxybutyrate; however, ASR extract reduces the 2-hydoxybutyrate, alanine,
allantoin, beta-glucose and N6-acetyllysine after eight weeks of daily therapy. In
serum, glycerol, 3-hydroxybutyrate, methionine and glucose were found higher in
diet-induced obesity rats compared to lean rats; however, serum glucose and glycerol
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were reduced after treated with ASR extract. Furthermore, score plots of partial least-
squares discriminant analysis showed 50% of obese rats treated with low dose ASR
extract was normalised. This finding is consistent with the result obtained from urine
and biochemical analyses of plasma, thus indicating that low dose ASR is more
effective than high dose in reversing lipid profile and glucose towards normal levels.
The lipid lowering effect of ASR extract might be contributed by ferulic acid, which
has the ability as HMG-CoA reductase inhibitor and leads to the decrement of LDL
cholesterol. The results of the present study demonstrates that low dose ASR extract
exhibited anti-hyperlipidaemia, leptin and glucose reversing effects of diet-induced
obesity rats, thus may be a useful therapeutic candidate for hyperlipidaemia.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Doktor Falsafah
PERUBAHAN METABOLIK PADA TIKUS OBES TERARUH DIET YANG
DIRAWAT DENGAN EKSTRAK ETANOL Alternanthera sessilis
Oleh
AZIZAH BINTI OTHMAN
Mei 2016
Pengerusi : Profesor Amin Ismail, PhD
Fakulti : Perubatan dan Sains Kesihatan
Alternanthera sessilis juga dikenali dengan nama keremak di Malaysia merupakan
tanaman tahunan yang tergolong dalam keluarga tanaman Amaranthaceae.
Penyelidikan masih kurang dijalankan ke atas spesis Alternanthera sessilis kerana
nilai komersialnya yang rendah. Kajian ini dijalankan untuk mengkaji komposisi
pemakanan, kapasiti antioksidan dan sebatian fenolik dalam ekstrak air dan etanol
Alternanthera sessilis merah (ASR) and Alternanthera sessilis hijau (ASG) yang di
tanam di Malaysia. Secara tradisional, ASR telah diambil sebagai minuman herba.
Walaubagaimanapun, terdapat sedikit sahaja data saintifik yang melaporkan tentang
kesan tumbuhan ini terhadap kesihatan. Justeru itu, kajian ini juga mengkaji kesan
pengambilan ekstrak ASR terhadap profil lipid, leptin, adiponektin, ujian fungsi hati
dan ujian fungsi ginjal pada plasma tikus obes teraruh diet. Sekiranya tikus obes
dirawat dengan ekstrak ASR, maka ia akan mengubah metabolit dalam urin dan
serum. Spektroskopi NMR bersama analisis multivariat telah digunakan untuk
mengkaji perubahan metabolit dan penanda biologi obesiti dalam urin dan serum
tikus obes teraruh diet. Hasil kajian menunjukkan bahawa ASR mempunyai
kandungan proksimat dan mineral signifikan (p <0.05) lebih tinggi berbanding ASG.
Kedua-dua ekstrak etanol ASR dan ASG menunjukkan kapasiti antioksidan lebih
tinggi berbanding ekstrak air secara signifikan <0.05. Jumlah kandungan fenolik
menunjukkan korelasi yang positif dengan kapasiti antioksidan yang mungkin
menjadi salah satu penyumbang kritikal kepada aktiviti antioksidan. Sebanyak
sebelas sebatian fenolik telah dikenalpasti dalam ekstrak ASR. Sebatian fenolik yang
tedapat dalam ekstrak ASR ialah asid galik, epigalokatekin, katekin, asid klorogenik,
asid 4-hidroksibenzoik, asid vanilik, asid ferulik, etil galat, merisetin, daidzin dan
apigenin. Ekstrak ASR pada dos rendah mempunyai penambahbaikan yang ketara
pada profil lipid dalam plasma serta menurunkan leptin selepas terapi setiap hari
selama lapan minggu. Manakala tikus obes yang dirawat dengan dos tinggi ekstrak
ASR mempunyai lemak viseral yang paling sedikit berbanding kumpulan lain.
Obesiti disebabkan oleh diet tinggi lemak menyebabkan peningkatan glukosa,
kreatinin, serotonin dan 3- hidroksibutirat dalam urin; bagaimanapun, ekstrak ASR
mengurangkan 2-hidroksibutirat, alanin, allantoin, beta-glukosa dan N6-asetillisina
selepas terapi setiap hari selama lapan minggu. Dalam serum, gliserol, 3-
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hidroksibutirat, metaionin dan glukosa didapati lebih tinggi dalam tikus obes teraruh
diet berbanding tikus kurus; bagaimanapun, glukosa dan gliserol dalam serum telah
berkurang selepas dirawat dengan ekstrak ASR. Tambahan pula, plot skor analisis
pembeza layan kuasa dua terkecil separa (PLS-DA) menunjukkan 50% daripada
tikus obes yang dirawat dengan ekstrak ASR pada dos rendah berada di kedudukan
yang sama dengan tikus kurus. Penemuan ini adalah konsisten dengan keputusan
yang diperolehi daripada urin dan ujian biokimia dalam plasma, justeru itu
menunjukkan bahawa ASR pada dos rendah adalah lebih berkesan daripada dos
tinggi pada kesan pembalikan profil lipid dan glukosa ke tahap normal. Kesan
penurunan lipid oleh ekstrak ASR berkemungkinan disebabkan oleh asid ferulik
yang berkebolehan menghalang HMG-CoA reduktase dan membawa kepada
penurunan kolesterol LDL. Keputusan kajian ini menunjukkan bahawa ekstrak ASR
dos rendah mempamerkan kesan anti-hiperlipidemia, menterbalikkan tahap leptin
dan glukosa ke arah normal dalam tikus obes teraruh diet, dengan itu boleh menjadi
sumber terapeutik berguna untuk hiperlipidemia.
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ACKNOWLEDGEMENTS
First and foremost, I am grateful to Allah S.W.T for having made this project
successful and for guiding me through the critical and difficult times.
This thesis would not have been possible without the help of so many people in so
many ways. I would like to express the deepest appreciation to my main supervisor,
Professor Amin Ismail for his guidance, patience, understanding, encouragement and
spending his precious time in helping me throughout my study. Special thanks also
dedicated to my co-supervisors, Assoc. Prof. Dr. Alfi Khatib and Assoc. Prof. Dr.
Barakatun Nisak Md Yusof for their support and advice.
Special appreciation dedicated to the laboratory staff from the Department of
Nutrition and Dietetics, Faculty Medicine and Health Sciences, UPM for their help
and guidance especially Mr. Syed Hasbullah, Mr. Simon, Mr Naim, Mr. Ramli, Mr.
Eddy and Ms Che Maznah; laboratory staff of Institute of Bioscience especially Mr.
Salahudin for his help throughout my work in NMR-based metabolomics. I would
especially like to thanks to all lecturers and staff at Faculty of Medicine and Health
Sciences, UPM.
I would also like to thank all my friends (Nurul Nadirah, Maulidiani, Azliana,
Hafeedza, Najla and Dr. Fuoad) who help me in animal study and multivariate data
analysis. My warmest thanks also goes to my husband, Mohd Zairi Haron; my
parents, Othman Mohammad and Zaharah Sulaiman; my sons (Muhammad Irfan and
Muhammad Ilman). Your prayers was what sustained me this far.
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Doctor of Philosophy.
The members of the Supervisory Committee were as follows:
Amin Ismail, PhD
Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Chairman)
Barakatun Nisak Md Yusof, PhD
Associate Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Member)
Alfi Khatib, PhD
Associate Professor
Kulliyah of Pharmacy
International Islamic University Malaysia
(Member)
_________________________________
BUJANG KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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Declaration by graduate student
I hereby confirm that:
this thesis is my original work;
quotations, illustrations and citations have been duly referenced;
this thesis has not been submitted previously or concurrently for any other degree
at any other institutions;
intellectual property from the thesis and copyright of thesis are fully-owned by
Universiti Putra Malaysia, as according to the Universiti Putra Malaysia
(Research) Rules 2012;
written permission must be obtained from supervisor and the office of Deputy
Vice-Chancellor (Research and Innovation) before thesis is published (in the form
of written, printed or in electronic form) including books, journals, modules,
proceedings, popular writings, seminar papers, manuscripts, posters, reports,
lecture notes, learning modules or any other materials as stated in the Universiti
Putra Malaysia (Research) Rules 2012;
there is no plagiarism or data falsification/fabrication in the thesis, and scholarly
integrity is upheld as according to the Universiti Putra Malaysia (Graduate
Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia
(Research) Rules 2012. The thesis has undergone plagiarism detection software.
Signature: _______________________ Date: __________________
Name and Matric No.: Azizah Binti Othman, GS30799
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Declaration by Members of Supervisory Committee
This is to confirm that:
the research conducted and the writing of this thesis was under our supervision;
supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate
Studies) Rules 2003 (Revision 2012-2013) are adhered to.
Signature: __________________
Name of
Chairman of
Supervisory
Committee: Professor Dr. Amin Ismail
Signature: __________________
Name of
Member of
Supervisory
Committee: Associate Professor Dr. Barakatun Nisak Md Yusof
Signature: __________________
Name of
Member of
Supervisory
Committee: Associate Professor Dr. Alfi Khatib
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TABLE OF CONTENTS
Page
ABSTRACT i
ABSTRAK iii
ACKNOWLEDGEMENTS v
APPROVAL vi
DECLARATION viii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF ABBREVIATIONS xii
CHAPTER
1 INTRODUCTION
1.1 Research Background 1
1.2 Problem Statement 2
1.3 Significance of Study 4
1.4 Objectives
1.4.1 General objective 5
1.4.2 Specific objectives 5
2 LITERATURE REVIEW
2.1 Alternanthera sessilis 6
2.2 Obesity and Chronic Diseases 11
2.3 Body Weight Management 12
2.3.1 Behaviour Change, Diet and Physical Activity 13
2.3.2 Treatment Drugs 15
2.3.3 Natural Products 17
2.4 Phenolic Compounds 18
2.5 Antioxidants 23
2.6 Metabolomics 24
3 COMPARATIVE EVALUATION OF NUTRITIONAL
COMPOSITIONS, ANTIOXIDANT CAPACITY AND
PHENOLIC COMPOUNDS OF Alternanthera sessilis
VARIETIES RED AND GREEN
3.1 Introduction 29
3.2 Materials and Method
3.2.1 Sample preparation 30
3.2.2 Preparation of extracts 30
3.2.3 Nutritional composition 31
3.2.4 Determination of total phenolic content 33
3.2.5 Extraction of phenolic compounds 34
3.2.6 Identification and quantification of phenolic
compounds
34
3.2.7 β-carotene bleaching assay 35
3.2.8 2,2-Diphenyl-2-picrylhydrazyl (DPPH) assay 35
3.2.9 2,2΄-Azinobis-3-ethylbenzothiazoline-6- 35
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sulphonic acid (ABTS) assay
3.2.10 Oxygen radical absorbance capacity (ORAC)
assay
36
3.2.11 Ferric reducing/antioxidant power (FRAP)
assay
36
3.3 Statistical Analysis 37
3.4 Results and Discussion
3.4.1 Nutrient compositions in ASR and ASG 37
3.4.2 Total phenolic content (TPC) 39
3.4.3 Phenolic compounds of ASR and ASG 40
3.4.4 β-carotene bleaching 43
3.4.5 ORAC value 44
3.4.6 Scavenging activity against DPPH radical 45
3.4.7 Scavenging activity against ABTS radical 45
3.4.8 Ferric reducing ability 46
3.5 Conclusion 46
4 PHYSIOLOGICAL AND BIOCHEMICAL EFFECTS OF
ETHANOLIC EXTRACT OF Alternanthera sessilis RED ON
HIGH FAT DIET-INDUCED OBESE RATS
4.1 Introduction 47
4.2 Materials and Methods
4.2.1 Plant material 48
4.2.2 Preparation of extract 48
4.2.3 Animals 48
4.2.4 Induction of obesity 49
4.2.5 Alternanthera sessilis red (ASR) extract
administration
52
4.2.6 Measurement of body weight and food intake 52
4.2.7 Blood and urine collection 52
4.2.8 Determination of plasma lipid profiles 52
4.2.9 Kidney and liver function test 53
4.2.10 Determination of plasma leptin and adiponectin 53
4.3 Statistical Analysis 53
4.4 Results and Discussion
4.4.1 Effect of high fat diet (HFD) on body weight of
male Sprague Dawley rats
53
4.4.2 Effect of treatment on body weight of obese
rats
55
4.4.3 Organs and visceral weights 58
4.4.4 Food intake 59
4.4.5 Biochemical parameters 59
4.5 Conclusion 67
5 METABOLITE CHANGES IN URINE AND SERUM OF
OBESE RATS SUPPLEMENTED WITH Alternanthera
sessilis EXTRACT BASED ON 1H NMR
5.1 Introduction 68
5.2 Materials and Methods
5.2.1 Plant material 68
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5.2.2 Animals 69
5.2.3 Induction of obesity 69
5.2.4 Alternanthera sessilis red (ASR) extract
administration
69
5.2.5 Experimental design 69
5.2.6 Preparation of urine samples and acquisition of
1H-NMR spectra
69
5.2.7 Preparation of serum samples and acquisition of
1H-NMR spectra
70
5.2.8 NMR data processing and statistical analysis 70
5.2.9 Multivariate statistical analysis 70
5.2.10 Models 71
5.3 Results and Discussion
5.3.1 1H NMR spectra of urine and serum obese and
lean rats
71
5.3.2 Multivariate pattern recognition analysis after
induction of obesity
76
5.3.3 Biomarkers for obese rats 82
5.3.4 Effect of ASR extracts on 1H NMR spectra of
urine and serum
86
5.3.5 Effect of diet modification on obese rat‘s
metabolites
86
5.3.6 Effect of ASR extracts on urine metabolites of
obese rats
91
5.3.7 Effect of ASR extracts on serum metabolites of
obese rats
98
5.3.8 Pathway analysis 103
5.4 Conclusion 108
6 SUMMARY, GENERAL CONCLUSION AND
RECOMMENDATION FOR FUTURE RESEARCH
109
REFERENCES 111
APPENDICES 131
BIODATA OF STUDENT 145
LIST OF PUBLICATIONS 145
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LIST OF TABLES
Table Page
2.1 Therapeutic effects of Alternanthera sessilis red (ASR) and
Alternanthera sessilis green (ASG)
9
2.2 Features of obesity-related dyslipidaemia 14
2.3 Anti-obesity drugs side-effects 16
2.4 Vegetables that possess anti-obesity 20
2.5 The advantages and limitation of NMR spectroscopy for
metabolomics research in comparison with MS spectrometry
25
3.1 Proximate analysis of Alternanthera sessilis red and green 38
3.2 Selected major and trace minerals of Alternanthera sessilis red and
green
38
3.3 Phenolic compounds identified by HPLC-ESI-MS 42
3.4 Total phenolic content and antioxidant capacity of ASR and ASG
extracts
44
4.1 Ingredients of high fat diet 49
4.2 The whole body composition analyses by dual-energy X- ray
absorptiometry (DEXA)
55
4.3 Effect of treatment on body weight of obese rats 57
4.4 Effect of treatment on organs and visceral weights 58
4.5 Food Intake of experimental rats during treatment period 59
4.6 Lipid profiles, leptin and adiponectin of lean and obese rats before
treatment (baseline).
60
4.7 Effect of treatment on biochemical parameters of obese rats 62
4.8 Effect of treatment on liver and kidney function of obese rats 66
5.1 Assignment of 1H chemical shift of metabolites observed in urine
DIO rats relatively to lean rats.
74
5.2 Assignment of 1H chemical shift of metabolites observed in serum
DIO rats relatively to lean rats.
75
5.3 Summary of significant urinary metabolites differentiating before
and after dietary changes of obese rats
90
5.4 PLS-DA and OPLS-DA Model Summary from Urine Sample.
92
5.5 Metabolite changes in urine identified by 1H NMR after treatment
relative to before treatment.
97
5.6 Metabolite changes in serum identified by 1H NMR after treatment
relative to before treatment.
102
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LIST OF FIGURES
Figure Page
2.1 Alternanthera sessilis green 7
2.2 Alternanthera sessilis red 7
2.3 Chemical structure of Orlistat 15
2.4 Chemical structure of Sibutramine 15
2.5 Chemical structure of phenol and polyphenol 18
2.6 General classification of phenolic compounds 21
2.7 Chemical structure of polyphenols 22
3.1 Freeze-dried Alternanthera sessilis red 31
3.2 Freeze-dried Alternanthera sessilis green 31
3.3 Total phenolic content of ASR and ASG extracts 39
3.4 HPLC chromatograms of phenolic compounds in Alternanthera
sessilis red and Alternanthera sessilis green
41
4.1 Preparation of high-fat 50
4.2 Obese rat with lean rat 50
4.3 Experimental design for animal study 51
4.4 Mean body weight of lean and obese rat before treatment 54
4.5 Body weight of obese and normal rats during 8 w of treatment 56
4.6 Lipid profiles, leptin and adiponectin changes (%) after 8 week
treated with ASR extracts
64
5.1 The overlaid 1H NMR spectra of urine samples obtained from DIO
and lean SD rats
72
5.2 The overlaid 1H NMR spectra of serum samples obtained from
DIO and lean SD rats
73
5.3 Principle component (PCA) obtained from 1H NMR spectra of
urine sample to compare the metabolome of the lean and obese rat
77
5.4 Principle component (PCA) obtained from 1H NMR spectra of
serum sample to compare the metabolome of the lean and obese rat
78
5.5 Partial least-squares discriminant analysis (PLS-DA) obtained from
1H NMR spectra of urine sample to compare the metabolome of the
lean and obese rat
79
5.6 Partial least-squares discriminant analysis (PLS-DA) obtained from
1H NMR spectra of serum sample to compare the metabolome of
the lean and obese rat
80
5.7 Orthogonal partial least-squares discriminant analysis (OPLS-DA)
score plot obtained from 1H NMR spectra of urine (A) and serum
(B) samples from obese and lean rats
81
5.8 Orthogonal partial least-squares discriminant analysis (OPLS-DA)
S-plot obtained from 1H NMR spectra of urine (A) and serum (B)
samples from obese and lean rats
83
5.9 OLPS-DA column loading plots of urine (A) and serum (B) sample 84
5.10 The overlaid 1H NMR spectra of urine (A) and serum (B) samples
obtained from control negative (Ob), control positive (Ob+Orlistat)
and Altenanthera sessilis treated rat‘s (Ob+ASRLC)
87
5.11 Score plot of partial least-squares discriminant analysis (PLS-DA)
obtained from 1H NMR spectra of urine sample to compare the
88
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metabolome week 0, week 4 and week 8 of non-treated obese rats
5.12 PLS weight of obtained from 1H NMR spectra of urine sample to
compare the metabolome week 0, week 4 and week 8 of non-
treated obese rats
89
5.13 PLS-DA Loading Column Plot with Variable Importance in the
Projection (VIP) Plot Score >1.
89
5.14 OPLS-DA scores plots (first row), S-plots (second row) and
coefficient loading plots (third row) obtained from different
treatment groups.
93
5.15 Partial least-squares discriminant analysis (PLS-DA) obtained from 1H NMR spectra of serum sample after treatment period.
99
5.16 PLS-DA loading column plots derived from NMR data for serum
samples
100
5.17 Comparison of serum metabolites between groups identified by 1H
NMR after treatment
104
5.18 MetPA analysis pathway of urine sample from group 175E 106
5.19 MetPA analysis pathway of serum sample from group 175E 106
5.20 Possible mechanism of ASR extract 107
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LIST OF ABBREVIATIONS
AAPH 2,2‘-azobis(2-amidino-propane)
ABTS 2,2΄-azinobis-3-ethylbenzothiazoline-6-sulphonic acid
ALT Alanine transaminase
ANOVA analysis of variance
ASG Alternanthera sessilis green
ASGEE Alternanthera sessilis green ethanolic extract
ASGWE Alternanthera sessilis green water extract
ASR Alternanthera sessilis red
ASREE Alternanthera sessilis red ethanolic extract
ASRWE Alternanthera sessilis red water extract
AST Aspartate transaminase
AUC area under the curve
BCB Beta carotene bleaching
BW Body weight
CMC Carboxymethylcellulose
CVD Cardiovascular disease
DAD Diode array detector
DIO Diet-induced obesity
DPPH 2,2-Diphenyl-2-picrylhydrazyl
ESI-MS Electrospray ionization mass spectrometry
FRAP Ferric reducing/antioxidant power
FW Fresh weight
GAE Gallic acid equivalents
HDL-C High density lipoprotein cholesterol
HFD High-fat diet
HPLC high performance liquid chromatography
IDL Intermediate-density lipoproteins
LC-MS Liquid chromatography–mass spectrometry
LDL-C Low density lipoprotein cholesterol
M-H+ / M-H- molecular ions
MW Molecular weight
NMR Nuclear magnetic resonance
OPLS-DA Orthogonal partial least-squares discriminant analysis
ORAC Oxygen radical absorbance capacity
PBS phosphate-buffer solution
PCA Principal component analysis
PDA photodiode array
PLS-DA Partial least squares discriminant analysis
SIM single ion monitoring
SIMCA Soft Independent Modeling of Class Analogy
SPSS Statistical Package for the Social Science
TC Total cholesterol
TE Trolox equivalents
TG Triglycerides
TPC Total phenolic content
TSP 3-Trimethylsilyl-propionic acid
VIP variable importance for the projection
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VLDL-C Very-low-density lipoprotein
w Week
WHO World Health Organisation
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CHAPTER 1
INTRODUCTION
Obesity is a major public health problem. The global prevalence of obesity has
doubled in the past 34 years, from 5% to 11% in men and from 8% to 15% in women
(WHO, 2015). Overall, more than 1.9 billion adults worldwide were overweight and
at least 600 million of them were obese (WHO, 2015). Overweight in adults is
defined as a body mass index (BMI) between 25.0 to 29.9; and BMI of 30 or higher
is considered obese. Recently, Malaysia has been rated as the highest among Asian
countries for obesity. Ng et al. (2014) stated that 49% of women and 44% of men in
this country were found to be overweight or obese (Ng et al., 2014). A national
survey among Malaysians aged 15 years and above reported that obesity prevalence
was higher in female compared to male; Malays and Indians had higher prevalence
of obesity compared with Chinese (Rampal et al., 2007). According to Aronne
(1998), excess body fat leads to a much higher risk of premature deaths and other
serious health problems including cardiovascular diseases, hypertension,
dyslipidaemia, stroke, diabetes mellitus, gallbladder disease, respiratory dysfunction,
gout, osteoarthritis and certain cancers.
Diet is a lifestyle factor that plays a major role in the primary and secondary
prevention of several chronic diseases including diabetes, cancer and stroke (WHO,
2012). Energy imbalance is the main cause for obesity, when energy intake is more
than energy expenditure. In order to maintain an ideal body weight, an individual
should take just the required amount of calories based on his/her energy expenditure.
Factors that influence energy expenditure are gender, growth, age, physical activity
and body composition. Our body uses energy to maintain basic physiological
function (basal metabolic rate), to do physical activity, and for adaptive
thermogenesis. Furthermore, the body‘s response to environmental temperature,
amounts and types of nutrient consumed (Smolin and Grosvenor, 2003). Indeed,
adaptive thermogenesis increases with the size of the meal. Therefore, to reduce
body weight, one should increase the total energy expenditure by reducing calorie
intake and/or increasing physical activity, which are difficult to accomplish by many
people.
Nowadays, natural diets have received more attention because of their beneficial
effects toward health as they contain antioxidant, anti-obesity, antimicrobial, anti-
inflammatory and anti-ageing properties. Increased consumption of fruits and
vegetables is associated with a lower risk of degenerative diseases (He et al., 2007).
Natural antioxidant substances are presumed to be safe since they occur in plant
foods, and are seen as more desirable than their synthetic counterparts. Data from
both scientific reports and laboratory studies show that plants contain a large variety
of phytochemical that possess antioxidant (Chanwitheesuk et. al., 2005). Typical
compounds that exhibit antioxidants include vitamins, carotenoids, and phenolic
compounds. Therefore, recommendations have been made to increase the daily
intake of fruit and vegetables, which are rich in these nutrients to lower the risk of
chronic health problems. Additionally, some of the vegetables are claimed to have
medicinal properties, such as prevention of ailments such as diabetes, high blood
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pressure, cardiovascular disease, arthritis, fever and coughs (Andarwulan et al.,
2010; Dauchet et al., 2010).
Alternanthera sessilis or ‗keremak‘ has been used for a variety of medicinal purposes
in Malaysia, India, Taiwan and Nigeria. There are two varieties of Alternanthera
sessilis: green, which commonly known as ponnankanni in Tamil (Thomas et. al.,
2014), and red, which is locally known as Huung Tien Wu in Chinese (Tan and Kim,
2013). Generally, the different colours are derived from different pigments.
Consequently, they influence their antioxidant capacity and bioactive compounds by
having different pigments as such carotene, lycopene and anthocyanins. The green
variety is eaten as vegetable to increase the supply of milk, used in treating night
blindness, relieving headache and dizziness, and for asthma (Lin et. al., 1994).
Whereas, the red variety is consumed as herbal drinks to lower blood cholesterol
level (Tan and Kim, 2013). In this study, obese rats were supplemented with
ethanolic extract of Alternanthere sessilis red (ASR) for 8 weeks. Supplementations
with ASR extract will alter the urine and serum metabolites towards normal state,
thus leads to improve the lipid profiles of obese rats.
1.2 Problem Statement
In Asia, especially in South East Asia, the prevalence of overweight among adults
range from 13.2% Indonesia to 32.5% Singapore (Khor, 2012). The highest
prevalence of adult obesity is in Singapore at 6.9%, compared to 2.4% in the
Republic of Korea and Indonesia (Low et al., 2009). According to Suzana et al.
(2012), the prevalence of overweight among Malaysian elderly population increases
from 16.6% to 29.1% between 1996 and 2006. Over the same period, the prevalence
of obesity in adults has increased three-fold, rising from 4.4% to 14%. Recently,
Malaysia has been rated as the highest among Asian countries for obesity with 17%
of the women and 11% of men in this country are obese (Ng et. al., 2014).
Obesity is often accompanied by high blood pressure, high blood cholesterol, type 2
diabetes and coronary heart disease (Panel, 1998). Although, genetics plays an
important role in the regulation of body weight in humans and animals, the
tremendous increase in prevalence of obesity worldwide cannot be explained well by
genetics. Dietary fat intake has often been claimed to be responsible for the increase
in body fat mass.
Drugs or sliming products are preferred by the public, compared to physical activity,
in order to reduce body weight instantly. An obesity drugs market is theoretically
huge, as it accounts up to 6% of total health care cost in several developed countries
(Birari and Bhutani, 2007). Existing obesity-treatment drugs on the market are
Sibutramine and Orlistat. Sibutramine is an appetite suppressant that inhibits the re-
uptake of noradrenaline and serotonin, promoting and prolonging satiety (Halford et
al., 2007). While Orlistat is an intestinal lipase inhibitor; it reduces 30 % intestinal
fat absorption through inhibition of pancreatic lipase. Both drugs have side-effects;
including increased blood pressure, dry mouth, constipation, headache, insomnia,
and cardiovascular disorder (Mohamed et al., 2014). For the reasons above, a wide
variety of natural materials have been explored for their obesity treatment potential.
.
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Alternanthera sessilis is among the indigenous plants in Malaysia that possess
medicinal properties based on traditional usage. However, no study has been reported
on the nutritional composition of Alternanthera sessilis. There were several chemical
constituents found in Alternanthera sessilis green (ASG) including cycloeucalenol,
choline, oleanolic acid, saponins, 5-a-stigmasta-7-enol and β-sitosterol (Borah et. al.,
2011; Ghani 2003; Rastogi and Mehrotra, 1998), while there has been no single
study reported on Alternanthera sessilis red (ASR). Therefore, the nutritional
composition and antioxidant capacity of local ASG and ASR are analysed in this
study.
Traditionally, ASG is used to treat cuts and wounds and skin diseases while it is an
antidote for snake bites and scorpion sting (Thomas et al., 2014) whereas the ASR
was believed to improve blood circulation and lower the cholesterol level (Tan and
Kim, 2013). Although the ASR is consumed as herbal drinks to reduce cholesterol, to
date, no scientific evidence has been published claiming the effects of ASR
supplements reducing cholesterol level. There have been several studies done that
investigated the health benefits of ASG including that they possessed antimicrobial,
antidiabetic, anti-inflammatory, antioxidant, hepatoprotective, and hematinic and
wound healing activities (Thomas et al., 2014; Arollado and Osi, 2010; Lin et al.,
1994). In contrast, only one study reported the effects of ASR on type 2 diabetic rats
(Tan and Kim, 2013), thus showing that this plant has not been extensively explored
yet, particularly regarding its effects on obesity. Therefore, the DIO animal model is
used to investigate the effect of 175 mg ASR extract/kg body weight and 350 mg
ASR extract/kg body weight to further examine this plant. The maximal dose 350
mg/kg BW and minimal dose of 175 mg/kg BW were chosen based on common
concentration used in anti-obesity and anti-diabetic studies of plant extracts (Ho et
al., 2012b; Sung et al., 2013).
Metabolomics has been used in the biomarker discovery of many diseases, such as
cardiovascular, diabetes and obesity (Wang et al., 2005; Roberts, et al., 2011). The
investigation of bio-fluids as such urine and plasma in obese models allows the
qualitative and quantitative determination of metabolites (Pelantová et al., 2015). In
addition, NMR-based metabolomics can be considered as a powerful method in
metabolomics research that allows for comprehensive metabolites profiling of bio-
fluid, which is non-destructive, and requires minimal sample preparation (Emwas et
al., 2013). Moreover, the result of metabolomics studies has demonstrated both
feasibility and flexibility across the physiological, pathological and epidemiological
human study (Du et al., 2013). The development of obesity, particularly DIO causing
metabolites alteration, thus could be determined by an NMR procedure.
The main aim of the present work is to investigate the effects of ASR extracts on
obese rats pertaining to physiological and biochemical parameters. However, a better
understanding on the metabolite changes is provided with metabolomics approaches.
Therefore, both biochemical and NMR-based metabolomics techniques were used to
discover the metabolite changes in the urine and serum obese rats after receiving
treatment of ASR extracts.
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1.3 Significance of Study
The rising prevalence of obesity in Malaysia and the parallel rise in the cost of health
care for obesity-treatment has propelled interest among researchers, as well as the
public, in seeking natural sources which are less expensive. Malaysia is one of the
countries that is blessed with rich but underutilised fruits and vegetables commonly
growing wild in the Peninsular Malaysia and East Malaysia (Sabah and Sarawak).
The underutilised fruits and vegetables contain high amounts of vitamins, minerals
and dietary fibres (Gupta et al., 2005). Fruits and vegetables are consumed because
of their taste, which adds variety and flavour to the Malaysian diet, as well as for
their health benefits (Lesschaeve and Noble, 2005)
In Malaysia, Alternanthera sessilis is rarely consumed, unfamiliar, and has not
received much attention compared to common vegetables and herbs. Based on
several previous studies, it was revealed that this plant has a potential application
towards health and disease prevention. Therefore, these plants are selected over the
other underutilised plants to provide scientific evidence, and consequently, diversify
its usage. There are two varieties available in Malaysia - the green (ASG) and red
(ASR). the nutritional values, nutritional qualities and antioxidant properties of ASG
(Kumar et al., 2011; Shyamala et al., 2005) while data on ASR have not been
reported yet. Therefore, the nutritional composition and antioxidant capacity
analyses for ASG and ASR were carried out in this study to discover their
nutritional values. The role of antioxidant in preventing diseases was reported by
numerous studies on both humans and animals (Zafra‐Stone et al., 2007; Willcox et
al., 2004; Vivekananthan et al., 2003; Brown et. al., 2001). Thus indicating that it is
important to analyse the antioxidant capacity in the studied samples.
Furthermore, results from this study could be used as reference to provide the
effect of low and high doses of ASR on obese rats on its physiological,
biochemical and metabolites, and thus can be used as a guide for other researcher.
This study is hoped to provide a better insight on the health benefits of
Alternanthera sessilis and to add value to this plant as part of Malaysian people‘s
diet.
A metabolomics method provides this research with an alternative to the traditional
single biomarker approaches to assess health and diseases. This kind of study will
be achieved by a maximum data capture from tissues or bio-fluids. The
metabolomics analysis could give a list of potential biomarkers of obese rats. In
addition to its benefit, this analysis also could provide a list of metabolic changes
due to the effects of treatment and dietary changes. Generally, weight loss program
will begin with diet modification, from high fat or high calorie diet to normal diet
along with other treatments. This was the reason why obese rats were given normal
diet instead of high fat diet. This study could reveal the effects of dietary changes
either with or without ASR supplementation on urine and serum metabolites. To
the best of our knowledge, there has been no published work on metabolomics
study of Alternanthera sessilis red variety in animals or human intervention.
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1.4 Objectives
1.4.1 General objective
To study the nutritional composition, antioxidant capacity and effects of
Alternanthera sessilis extracts on diet-induced obesity in rats.
1.4.2 Specific objectives
1. To determine the nutritional composition and antioxidant capacity of
Alternanthera sessilis red (ASR) and Alternanthera sessilis green (ASG)
2. To evaluate the effects of low and high doses of ASR extracts on physiological
and biochemical parameters in the plasma of diet-induced obese rats.
3. To investigate the metabolite changes of diet-induced obese rats after eight
weeks of being supplemented with low and high doses of ASR extracts using
NMR coupled with multivariate data analysis.
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APPENDICES
Appendix A: Standard Curve
1. Iron (II) sulphate heptahydrate (FeSO4. 7H2O) standard curve for the
determination of FRAP value
2. Gallic acid standard curve for the of total phenolic content
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3. Standard curve for the determination of leptin
4. Standard curve for the determination of adiponectin
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Appendix B: LC-ESI-MS spectra
1. LC-ESI-MS spectra acquired in negative and positive full-scan mode
corresponding to the [M-H]- and[M+H]
+ ions of each identified phenolic acid
extracted from the Alternanthera sessilis red.
(-) 4-hydroxybenzoic acid
(-) Apigenin
(-) Catechin
(+) Catechin
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(-) Chlorogenic acid
(-) Ferulic acid
(-) Gallic acid
(-) EGC
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(+) EGC
(-) myricetin
(+) myricetin
(-) Ethyl gallate
(+) Daidzein
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(-) Vanillic acid
(-) Ferulic acid
(-) Chlorogenic acid
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2. LC-ESI-MS spectra acquired in negative and positive full-scan mode
corresponding to the [M-H]- and[M+H]
+ ions of each identified phenolic acid
extracted from the Alternanthera sessilis green.
(-) 4-hydroxybenzoic
(-) Catechin
(+) Catechin
(+) Chlorogenic acid
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(-) EGC
(+) EGC
(-) Ethyl gallate
(-) 4-hydroxybenzoic
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(-) Apigenin
(-) Ferulic acid
(+) Vanillic acid
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Appendix C: S-plot (OPLS-DA)
1. S-plot urine final (3F vs 5F)
2. S-plot urine final (3F vs 1F)
5F 3F
3F 1F
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3. S-plot urine final (2F vs 3F)
4. S-plot urine final (3F vs 4F)
2F 3F
3F 4F
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Appendix D: Loading scatter plot
1. Loading scatter plot serum final (PLS-DA)
2. Loading scatter plot serum final (PCA)
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3. Loading scatter plot urine final (PLS-DA)
4. Loading scatter plot urine final (PCA)
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Appendix E: Ethics Approval
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BIODATA OF STUDENT
The student was born on 6th April 1980 in Kuala Kangsar, Perak, Malaysia. She
received her Bachelor of Science (Nutrition and Community Health) and Master of
Science (Nutritional Biochemistry) and from Universiti Putra Malaysia (UPM) in the
year 2002 and 2006, respectively. On August 2007, she was offered as lecturer in
Department of Food Technology, Faculty of Applied Sciences, Universiti Teknologi
MARA, Shah Alam, Malaysia. She has taught Nutrition for 4 Years before going on
study leave to pursue her PhD in Nutritional Sciences also at UPM.
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LIST OF PUBLICATIONS
Othman, A., Ismail, A., Hassan, F. A., Yusof, B. N. M., & Khatib, A. (2016).
Comparative evaluation of nutritional compositions, antioxidant capacities,
and phenolic compounds of red and green sessile joyweed (Alternanthera
sessilis). Journal of Functional Foods, 21, 263-271.
Azizah, O., Amin, I. and Barakatun Nisak, M. Y. (2014). Lipid lowering effect of
Alternanthera sessilis red. Obesity Review, 15 (suppl. 2), 129-176.
Abstract of the 12th
International Congress on Obesity, 17-20 March
2014, Kuala Lumpur, Malaysia (Poster presentation).
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UNIVERSITI PUTRA MALAYSIA
STATUS CONFIRMATION FOR THESIS / PROJECT REPORT AND COPYRIGHT
ACADEMIC SESSION :
TITLE OF THESIS / PROJECT REPORT : METABOLIC CHANGES IN DIET-INDUCED OBESITY AMONG RATS TREATED WITH ETHANOL EXTRACT OF Alternanthera sessilis RED NAME OF STUDENT : AZIZAH BINTI OTHMAN
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belonged to Universiti Putra Malaysia and I agree to allow this thesis/project report to be placed at
the library under the following terms: 1. This thesis/project report is the property of Universiti Putra Malaysia.
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purposes only.
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exchange. I declare that this thesis is classified as : *Please tick (√ )
CONFIDENTIAL (Contain confidential information under Official Secret
Act 1972).
RESTRICTED (Contains restricted information as specified by the organization/institution where research was done).
OPEN ACCESS I agree that my thesis/project report to be published
as hard copy or online open access. This thesis is submitted for :
PATENT Embargo from_____________ until ______________ (date) (date)
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