PREVENTIVE ROLE OF α-LIPOIC ACID, CURCUMIN AND GINGER

122 Pages·2014·1.64 MB·English

by  Hardik Ghelani

PREVENTIVE ROLE OF α-LIPOIC ACID, CURCUMIN AND
GINGER IN INHIBITING MYOGLOBIN GLYCATION AND
THE FORMATION OF ADVANCED GLYCATION END
PRODUCTS (AGEs)

Hardik Ghelani
(17601883)
(BPharm, MPharm (Pharmacology)

A thesis submitted in fulfilment of the requirements for the degree of
Master of Science (Honours)

School of Science and Health,
University of Western Sydney
October, 2014

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STATEMENT OF AUTHENTICITY

This thesis is submitted in fulfilment of the requirements for the postgraduate research
degree, Master of Science (Hon.), at the School of Science and Health, University of
Western Sydney. The work presented in this thesis is, to the best of my knowledge and
belief, original except as acknowledged in the text. I hereby declare that I have not
previously submitted this material, either in whole or part at any other institution.

_____________________
HARDIK GHELANI

Date: 3rd October-2014

Place: Campbelltown, NSW, Australia
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LIST OF PUBLICATIONS
 Ghelani, H.S., Razmovski-Naumovski, V., Kyada, A.V., Parekh, H.S., Li, G.Q., &
Nammi, S. (2014) Myoglobin and Low Density Lipoprotein Glycation in Cardiac
Complications of Diabetes: Current Perspectives and Therapeutic Options. Indian
Journal of Medical Research (In Press).
 Ghelani, H.S., Razmovski-Naumovski, & Nammi, S. (2014). Ginger inhibits
glucose and fructose-induced myoglobin glycation and advanced glycation end
products formation in vitro. Manuscript under preparation for submission to BMC
Complementary & Alternative Medicine.
 Ghelani, H.S., Razmovski-Naumovski, & Nammi, S. (2014). α-lipoic acid, a new
atici-glycation agent inhibits glucose and fructose-induced myoglobin glycation in
vitro. Manuscript under preparation for submission to Basic & Clinical
Pharmacology & Toxicology.
 Ghelani, H.S., Razmovski-Naumovski, & Nammi, S. (2014). Curcumin prevents
AGEs formation and protein oxidation against glucose and fructose-induced
myoglobin glycation in vitro. Manuscript under preparation for submission to
Journal of Pharmacy & Pharmacology.

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AKNOWLEDGEMENTS
Each morning comes with new opportunities in the form of numerous rays of hope.
Choosing a single ray, I decided to carry out something novel which would prove to be
fruitful and beneficial to me and society. It would be a great reward for me if my work
proves to help an endless number of patients. But, it’s not at all possible to walk alone on
this path of research, and it requires a pool of genuine, authentic, brilliant, elegant hands to
be by my side and I take this opportunity to oblige honour and pay gratitude to one and all. I
pay my utmost gratitude to my supervisor, Dr Srinivas Nammi for being a constant
deliverer of hands in the hour of need. I owe him the full share for the successful completion
of my project. I am thankful to him for providing the liberations in correct manner, for
valuable suggestions, for constructive criticism, for all inspiration that he gave me. Without
his guidance, my project would not have seen the ending shores. I take this opportunity to
thank my co-supervisor, Dr Valentina Naumovski for her timely suggestions, generous and
friendly nature, persistent encouragement, critical remarks and counsel during the whole
course of this work. I would like to acknowledge the instructional and handy tips of Mr
Ashish Kyada at the right time. I owe a lot to him for his valuable suggestions, constant
moral support and timely help throughout this project. I sincerely wish to acknowledge the
invaluable technical support extended by the laboratory technicians of the School, Mrs
Kathleen Kyle for her constant help and providing the requirements at the right time for
carrying out the experiments. Finally, I would like to mention my family, especially my
parents and my cousin brother Mr Vipul Ghelani, who have given moral, emotional and
financial support, and have great trust on my academic career.

- HARDIK GHELANI

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To
Science

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Table of Contents Page No
Cover page……………………………………………………………………………………………..1
Statement of authentication……………………………………………………………………….......2
List of publications…………………………………………………………………………………….3
Acknowledgements...............................................................................................................................4
Table of contents...................................................................................................................................6
List of tables…………………………………………………………………………………………...9
List of figures………………………………………………………………………………………...10
General abbreviations.........................................................................................................................11
Summary.............................................................................................................................................12
CHAPTER – 1 INTRODUCTION……………………………………………........14
1.1 Pathophysiology of hyperglycaemia-induced biochemical alteration: Focus on advanced
glycation end products………………………………………………………………………...15
1.1.1 Non-enzymatic glycation and advanced glycation end-products ………………….17
1.1.2 Fructose-induced protein glycation…………………………………………….......21
1.1.3 Receptors for advanced glycation end products…………………………………….23
1.1.4 Advanced glycation end products and heart failure………………………………...23
1.1.5 Myoglobin and its physiological role in the heart ……………………………........25
1.1.6 Myoglobin glycation and its possible role in diabetic cardiovascular
complications……………………………………….................................................26
1.2 Therapeutics strategies to inhibit protein glycation and advanced glycation end products
formation………………………………………………………………………………………30
1.2.1 Glycaemic control…………………………………………………………………..30
1.2.2 Synthetic AGEs inhibitors………………………………………………………….31
1.2.3 Natural AGEs inhibitors………………………………………………………........33
1.2.4 Antioxidants………………………………………………………………………...35
1.3 Prospects of alternative medicine therapy…………………………………………………….37
1.3.1 Overview………………………………………………………………………........37
1.3.2 α-Lipoic acid…………………………………………………………………..........37
1.3.3 Curcumin……………………………………………………………………….......38
1.3.4 Ginger……………………………………………………………………………….40
1.4 Rationale and objectives...…………………………………………………………………….42
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CHAPTER – 2 MATERIALS AND METHODS…………………………………..44
2.1 Chemicals………………………………………………………………………………........45
2.2 Equipment…………………………………………………………………………………...45
2.3 Preparation of reagents……………………………………………………………...............45
2.4 The test substances and dosage selection……………………………………………………49
2.5 Evaluation of myoglobin glycation inhibitory effect of α-lipoic acid, curcumin and ginger
under glucose/fructose overload in vitro…………………………………………………….51
2.5.1 Estimation of total fluorescent advanced glycation end products…………….51
2.5.2 Estimation of liberated free iron (ferrozine test)………………………………..52
2.5.3 Estimation of fructosamine (glycated myoglobin)……………………………...54
2.5.4 Estimation of protein carbonyls…………………………………………………55
2.5.5 Estimation of protein thiols………………………………………………........56
2.5.6 Data and statistical analysis…………………………………………………….57

CHAPTER – 3 RESULTS…………………………………………………………..58
3.1 Effect of α-lipoic acid, curcumin and ginger on myoglobin glycation-induced formation of
fluorescent advanced glycation end products……………………………………………........59
3.2 Effect of α-lipoic acid, curcumin and ginger on myoglobin glycation-mediated liberated free
iron…………………………………………………………………………………………….65
3.3 Effect of α-lipoic acid, curcumin and ginger on myoglobin glycation-induced fructosamine
formation………………………………………………………………....................................71
3.4 Effect of α-lipoic acid, curcumin and ginger on myoglobin glycation-induced protein
carbonyls formation ………………………………………......................................................77
3.5 Effect of α-lipoic acid, curcumin and ginger on myoglobin glycation-induced protein thiols
oxidation………………………………………........................................................................83
CHAPTER – 4 DISCUSSION………………………………………………….......89
4.1. Anti-glycation activity of α-lipoic acid……………………………………………………...93
4.2. Anti-glycation activity of curcumin…………………………………………………………96
4.3. Anti-glycation activity of ginger extract………………………………………………….....98

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CHAPTER – 5 CONCLUSION AND FUTURE DIRECTION………..........................102
5.1. General conclusion……………………………………………………………………........103
5.2. Limitations…………………………………………………………………………….........104
5.3. Future direction…………………………………………………………………………….105

BIBLIOGRAPHY…………………………………………………………..........106
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LIST OF TABLES
Table
Chapter Title of the table Page No.
No.
1.1 1 Effects of oral hypoglycaemic agents in glycaemic control 31
Some important synthetic compounds/
1.2 1 34
phytoconstituents with anti-glycation activity
Mechanism of action and clinical significance of known natural
1.3 1 36
antioxidants
2.1 2 Pharmacological activities of the selected test compounds 50
Effect of α-lipoic acid, curcumin and ginger on the formation of
3.1 3 62
fluorescent AGEs in myoglobin-glucose glycation.
Effect of α-lipoic acid, curcumin and ginger on the formation of
3.2 3 63
fluorescent AGEs in myoglobin-fructose glycation.
Effect of α-lipoic acid, curcumin and ginger on free iron release
3.3 3 68
in myoglobin-glucose glycation
Effect of α-lipoic acid, curcumin and ginger on free iron release
3.4 3 69
in myoglobin-fructose glycation
Effects of α-lipoic acid, curcumin and ginger on fructosamine in
3.5 3 74
myoglobin-glucose glycation
Effects of α-lipoic acid, curcumin and ginger on fructosamine in
3.6 3 75
myoglobin-fructose glycation
Effects of α-lipoic acid, curcumin and ginger on carbonyl
3.7 3 80
content in myoglobin-glucose glycation
Effects of α-lipoic acid, curcumin and ginger on carbonyl
3.8 3 81
content in myoglobin-fructose glycation
Effects of α-lipoic acid, curcumin and ginger on the level of thiol
3.9 3 86
group in myoglobin-glucose glycation
Effects of α-lipoic acid, curcumin and ginger on the level of thiol
3.10 3 87
group in myoglobin-fructose glycation

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LIST OF FIGURES
Fig
Chapter Title of the figures Page No.
No.
1.1 1 Formation of AGEs through various biochemical steps 20
Mechanism of fructation for the production of reactive carbonyl
1.2 1 22
and oxygen species
1.3 1 Fructose autoxidation 22
1.4 1 Summary of the AGEs pathways which may cause heart failure. 26
Possible role of myoglobin glycation in diabetes-induced
1.5 1 29
myocardial damage
1.6 1 α-Lipoic acid 38
1.7 1 Curcumin 40
1.8 1 Major bio-active phytoconstituents of ginger 41
Percentage inhibition of fluorescent AGEs formation by α-lipoic
acid (A), curcumin (B) and ginger (C) in myoglobin-glucose
3.1 3 64
(Mb-Glu) and myoglobin-fructose (Mb-Fru) glycation after 30
days of incubation.
Percentage inhibition of free iron release by α-lipoic acid (A),
curcumin (B) and ginger (C) in myoglobin-glucose (Mb-Glu)
3.2 3 70
and myoglobin-fructose (Mb-Fru) glycation after 30 days of
incubation.
Percentage inhibition of fructosamine formation by α-lipoic acid
(A), curcumin (B) and ginger (C) in myoglobin-glucose (Mb-
3.3 3 76
Glu) and myoglobin-fructose (Mb-Fru) glycation after 30 days of
incubation.
Percentage inhibition of protein carbonyl content by α-lipoic acid
(A), curcumin (B) and ginger (C) in myoglobin-glucose (Mb-
3.4 3 82
Glu) and myoglobin-fructose (Mb-Fru) glycation after 30 days of
incubation.
Percentage protection of protein thiol by α-lipoic acid (A),
curcumin (B) and ginger (C) in myoglobin-glucose (Mb-Glu)
3.5 3 88
and myoglobin-fructose (Mb-Fru) glycation after 30 days of
incubation.
Schematic representations of pathways induced by myoglobin
4.1 4 glycation and the inhibitory effects of α-lipoic acid, curcumin 101
and ginger extract on this pathway

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