antioxidant activity and hepatoprotective potential of leaf extracts from morella serrata

145 Pages·2017·3.94 MB·English

ANTIOXIDANT ACTIVITY AND HEPATOPROTECTIVE
POTENTIAL OF LEAF EXTRACTS FROM MORELLA SERRATA
(LAM.) KILLICK (MYRICACEAE).

By

Mbhele, Nobuhle

A dissertation submitted in accordance with the requirements for the Magister
Scientiae degree in the Faculty of Natural and Agricultural Sciences, Department of
Plant Sciences at the University of the Free State, QwaQwa Campus.

Supervisor: Dr. Anofi O.T Ashafa
Co- supervisors: Dr. Hafiz A. Abdelgadir and Dr. Ashwell R. Ndhlala

DECEMBER, 2016
Supervisor:
Dr. Anofi O.T Ashafa
Phytomedicine and Phytopharmacology Research Group
Department of Plant Sciences
University of the Free State Qwaqwa campus
Private Bag X13
Phuthaditjhaba
9866

Co-supervisor:
Dr. Hafiz A. Abdelgadir
Crop Science division
Agro-processing of medicinal plants
Agricultural Research Council- Vegetable and Ornamental Plants (VOP)
Private Bag X293
Pretoria
0001

Co- supervisor:
Dr. Ashwell R. Ndhlala
Crop Science division
Agro-processing of medicinal plants
Agricultural Research Council- Vegetable and Ornamental Plants (VOP)
Private Bag X293
Pretoria
0001
DECLARATION
Research title: Antioxidant activity and hepatoprotective potential of leaf extracts
from Morella serrata (Lam.) Killick (Myricaceae).

I, Mbhele Nobuhle (Student number: 2009131158), do hereby declare that the
dissertation hereby submitted for the qualification for the degree Master Scientiae in
Botany at the University of the Free State represents my own original, independent work
and that I have not previously submitted the same work for a qualification at another
university.

I further cede copy right of the dissertation in favour of the University of the Free State.

Signature: …………………………..

Date: ……………………………….

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DEDICATION
To my late father Thamsanqa Jerom Mbhele.

“You left so soon dad, you know how you left us and it pains me deeply to
think that you are not here anymore to share grateful times with me. I know
you are looking back on us. You will always be loved and missed”.

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ACKNOWLEDGEMENTS
Firstly, I would like to thank the Almighty God for leading, guiding and giving me unbelievable
power when I needed it the most.

To my main supervisor Dr. Anofi Omatoyo Tom Ashafa, Acting Dean of Faculty of Natural and
Agricultural Sciences, University of the Free State, Qwaqwa campus for his constant help,
valuable guidance, pressure, constructive suggestions and most importantly father figure role you
played in my life. Thank you for being a father to me. I will forever be grateful of meeting you in
my life.

To my Co-supervisors Dr. Hafiz Abdelgadir and Dr. Ashwell Ndhlala from the Agricultural
Research Council, thank you so much for such an opportunity you have given me. Thank you for
your support and guidance throughout my studies.

My deep gratitude goes to two PhD students, Mr Fatai Balogun and Sabiu Saheed. Thank you for
being a source of support and guidance. I thank God that I have met such wonderful souls during
the period of my study and I will forever be grateful for that.

Miss Getrude Mahanke, thank you for your constant help sister. I will always remember the
laughter we shared during the course of the study. Thank you so much and full of appreciation.

To PpRg members thank you all for the support, great efforts and sacrificing your time in the
fulfilment of this work.

To Agro-processing members at the Agricultural Research Council- Vegetable and Ornamental
Plants, I am very grateful that I have found sisters and brothers within you.

To my husband Bonga Njoko, thank you for your patience, love, support and encouragement
everything else. Thank you my life partner.

To the lady who saw a potential in me, Miss Vezi. Thank you so much for your help and
believing in me. I would not be where i am today if it wasn’t for you. Thank you.

Finally, I would like to acknowledge the Agricultural Research Council and National Research
Foundation for funding my studies.

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RESEARCH OUTPUTS
Conference proceedings
Mbhele, N., Abdelgaldir, H.A., Ndhlala, A.R., Ashafa, A.O.T., 2016. Phytochemical
analysis and antioxidant activity of leaf extracts of Morella serrata (Lam.) Killick
(Myricaceae). 42nd Annual Conference of the South African Association of Botanists
(SAAB) and the 12th Southern African Society for Synthetic Biology (SASSB), 10 – 13
January 2016. University of the Free State, Bloemfontein.

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TABLE OF CONTENTS
DECLARATION ....................................................................................... i
DEDICATION ........................................................................................... ii
ACKNOWLEDGEMENTS ...................................................................... iii
RESEARCH OUTPUTS ........................................................................... iv
TABLE OF CONTENTS ............................................................................v
LIST OF FIGURES .................................................................................. xi
LIST OF TABLES .................................................................................. xiii
ABBREVIATIONS AND SYMBOLS .................................................... xiv
LIST OF UNITS...................................................................................... xvi
ABSTRACT ........................................................................................... xvii
GRAPHICAL ABSTRACT .................................................................... xix

CHAPTER 1: General introduction ..........................................................1
1.1 Background of the study ............................................................................................ 1
1.2 Problem statement and justification of the study ........................................................ 3
1.3 Aim and objectives of the study ................................................................................. 5
1.4 Hypothesis ................................................................................................................. 6
1.5 Significance of the study ............................................................................................ 7
1.6 Outline of the dissertation .......................................................................................... 7
References…………………………………………………………………………………9

CHAPTER 2: Literature review: An overview on free radicals, oxidative
stress, antioxidant and Carbon tetrachloride-induced liver damage .... .13
2.1 Free radicals ........................................................................................................... .13
2.1.1 Reactive oxygen species and Reactive nitrogen species............................14
2.1.2 Sources of free radicals.................................................................................14
2.1.3 Beneficial and deleterious effects of RONS..............................................15
2.1.4 Oxidative stress and nitrosative stress........................................................15
2.1.5 Prevalence of pathological conditions associated with free radicals and
Oxidative stress worldwide and in South Africa...................................................16
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2.2 Antioxidants...............................................................................................................17
2.2.1 Natural antioxidants....................................................................................18
2.2.2 Natural antioxidant versus synthetic antioxidants......................................20
2.3 Secondary metabolites……………………………………………………………...21
2.4 Liver………………………………………………………………………………...21
2.4.1 Liver diseases..............................................................................................23
2.4.1.1 Prevalence of liver diseases.........................................................24
2.4.1.2 Limitations of treatments of liver diseases..................................24
2.4.1.3 Medicinal plants as a cure for liver diseases...............................24
2.5 Hepatotoxicity……………………………………………………………………...25
2.5.1 Hepatotoxin: Carbon tetrachloride (CCl )..................................................26
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2.5.1.1 Mechanism of Hepatotoxicity caused by carbon tetra-chloride in
the liver....................................................................................................26
2.6 Antihepatotoxin: Silymarin………………………………………………………....27
2.7 Medicinal plants…………………………………………………………………….28
2.7.1 The use, threats and conservation means of medicinal plants in South
Africa...................................................................................................................28
2.8 Choice of plant of study: Morella serrata (Lam.) Killick (Myricaceae)...................29
2.8.1 Classification of Morella serrata................................................................29
2.8.2 M. serrata common names.........................................................................30
2.8.3 Botany of M. serrata..................................................................................30
2.8.4 Geographical distribution of M. serrata.....................................................31
2.8.5 Threat to M. serrata....................................................................................31
2.8.6 Traditional uses of M. serrata....................................................................31
2.8.6.1 Medicinal uses.............................................................................31
2.8.6.2 Non-medicinal uses.....................................................................32
2.8.7 Phytochemical and pharmacological review of M. serrata........................32
References.......................................................................................................................33

CHAPTER 3: Qualitative and quantitative analysis of leaf extracts from
Morella serrata................................................................................46
3.1 Introduction.............................................................................................................46
3.2 Material and methods..............................................................................................46
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3.2.1 Collection, identification and authentication of plant material................46
3.2.2 Chemicals and reagents...........................................................................47
3.2.3 Preparation of crude extracts...................................................................47
3.2.4 Qualitative analysis of leaf extracts from M. serrata…………………...47
3.1.4.1 Preliminary phytochemical screening of M. serrata.................47
Detection of alkaloids................................................................47
Detection of tannins...................................................................47
Detection of phlabotannins........................................................47
Detection of saponins................................................................47
Detection of flavonoids..............................................................48
Detection of steroids..................................................................48
Detection of terpenoids..............................................................48
Detection of cardiac glycosides.................................................48
Detection of resins.....................................................................48
Detection of phenols..................................................................48
3.2.5 Quantitative determination of leaf extracts of M. serrata........................48
3.2.5.1 Preparation of standard solution and test concentration............48
3.2.5.2 Total phenolic content, total flavonoid content and total flavonol
content determination............................................................................49
3.2.6 Statistical analysis.....................................................................................50
3.3 Results......................................................................................................................50
3.4 Discussion.................................................................................................................56
3.5 Conclusions...............................................................................................................59
References.......................................................................................................................60

Chapter 4: In vitro antioxidant and free radical scavenging activity of
Morella serrata leaf
extracts......................................................................................................65

4.1 Introduction..............................................................................................................65
4.2 Material and methods...............................................................................................65
4.2.1 Collection, identification and authentication of plant material..................65
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4.2.2 Chemicals and reagents..............................................................................65
4.2.3 Preparation of crude extracts......................................................................66
4.2.4 Preparation standard solutions and test concentrations..............................66
4.2.5 In vitro antioxidant assays..........................................................................67
4.2.5.1 DPPH radical scavenging activity...............................................67
Principle of the method...............................................................67
Protocol.......................................................................................68
4.2.4.2 ABTS free radical scavenging activity........................................69
Principle of the method...............................................................69
Protocol.......................................................................................70
4.2.5.3 Nitric oxide scavenging activity..................................................71
Principle of the method...............................................................71
Protocol.......................................................................................71
4.2.5.4 Hydroxyl radical scavenging activity..........................................72
Principle of the method...............................................................72
Protocol.......................................................................................72
4.2.5.5 Reducing power capacity............................................................72
Principle of the method...............................................................72
Protocol.......................................................................................73
4.2.5.6 Hydrogen peroxide activity.........................................................73
Principle of the method...............................................................73
Protocol.......................................................................................74
4.2.5.7 Metal chelating ability.................................................................74
Principle of the method...............................................................74
Protocol.......................................................................................74
4.2.6 Statistical analysis......................................................................................75
4.3 Results.......................................................................................................................75
4.4 Discussion..................................................................................................................82
4.5 Conclusions................................................................................................................84
References........................................................................................................................85
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