Evaluation of the antioxidant and in vitro cytotoxic activity of Averrhoa carambola, Aegle marmelos

106 Pages·2015·5.05 MB·English

Evaluation of the antioxidant and in vitro cytotoxic activity
of Averrhoa carambola, Aegle marmelos and Spondias
pinnata leaf extracts

A DISSERTATION SUBMITTED TO BRAC UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIRMENTS FOR
THE MASTER OF SCIENCE IN BIOTECHNOLOGY

Submitted by-
Anahita Tanzia Zaman
Student ID: 11276010
April 2015

Biotechnology Program
Department of Mathematics and Natural Sciences
BRAC University
Bangladesh
Dedicated
To
My amazing parents
DECLARATION
The research work embodying the results reported in this thesis entitled “Evaluation of the
antioxidant and in vitro cytotoxic activity of Averrhoa carambola L, Aegle marmelos and
Spondias pinnata leaf extracts” submitted by Anahita Tanzia Zaman in partial fulfillment of the
requirements for the degree of Master of Science in Biotechnology, Department of Mathematics
and Natural Science, BRAC University, Dhaka. It is further declared that the research work
presented here is original and has not been submitted to any other institution for any degree or
diploma. The work was performed at Laboratories of BRAC University, The University of
Dhaka and The University of Asia Pacific, Dhaka, Bangladesh.

Candidate

_______________________
Anahita Tanzia Zaman

Supervisor Supervisor

________________________ _________________________

Dr. Naiyyum Choudhury Dr. Chowdhury Rafiqul Ahsan

Co-ordinator, MS in Biotechnlgy Professor

Department of Mathematics and Department of Microbiology
Natural Sciences

Brac University University of Dhaka
Mohakhali, Dhaka Dhaka
Acknowledgement

First and foremost, I express my deepest gratitude to almighty Allah for endowing me with health,
patience, protection and faith in all aspect of my life. During the time of my study I have worked with
many people for whom I have great regard, and I wish to extend my warmest thanks to all those who have
helped me to complete the thesis.

I am overwhelmed to express my respect, sincere gratitude and heartfelt thanks to Dr. Chowdhury Rafiqul
Ahsan, Professor, Department of Microbiology, University of Dhaka for his inspiration, constructive
criticism, affectionate guidance, permissions to work in various laboratories in order to get my work done,
positive attitude and specially for his scholastic knowledge in helping me to complete my thesis paper.
Without him, my study and research would not be complete. I owe him my deepest and utmost gratitude.

I would like to convey my indebtedness to Professor Naiyyum Chowdhury, Coordinator, Biotechnology
and Microbiology, Department of Mathematics and Natural Sciences, BRAC University, for his
inspiration, prudent advice, and immense patience in guiding me and for giving me the opportunity to
work whole heartedly.

I am grateful to Professor A A Ziauddin Ahmad, Chairperson, MNS department for allowing me to
pursue my post graduate studies in the department of MNS and for his constant guidance and help
throughout my entire period of study in the department.

I would like to convey my heightened appreciation to all my respected teachers of the Department of
Mathematics and Natural Sciences, BRAC University, for their academic counsel and encouragement.

I would particularly like to express my deepest thanks to Mr. Shashwata Biswas who provided me with
constant knowledge, his valuable time and tolerance towards my work, sound advice and good company
throughout the time of my the study. I gratefully acknowledge him for all that he has done for me.

I owe a debt of gratitutde to Mohammad Shahriar, Assistant Professor, Department of Pharmacy,
University of Asia Pacific, Dhanmondi, for allowing me to work in his laboratory, use their services with
complete comfort and helping me complete my work in peace. Also, I owe a great debt of gratitude to
Sheikh Ariful Hoque, Ph.D, Senior Scientist, CARS, University of Dhaka, for his complete guidance,
knowledge and opening his laboratory and allowing me to work with specialized equipment’s without
which my work would remain incomplete.

I am also grateful to the people of each of the laboratories I have worked in. I thank them from the bottom
of my heart for their support, and concerns. I am grateful to my loving friends, who constantly
encouraged me and pushed me to get through whenever I felt like giving up.

Finally, I have no words to thank my Mother, who made me get admission for my Master’s Program
before she departed, her love and blessings are still upon me. My Father, without whom I wouldn’t be
here where I stand today, I thank him for his love, care, encouragement, the biggest pillar of support
anyone could ever give me and for always being there, my brother, who never gave up supporting me
throughout my time of research and my family members for their unconditional love and support through
all the hassles and unavoidable circumstances and helping me pull through with ease.

The Author
Department of Mathematics and Natural Sciences
BRAC University, April 2015.
ABSTRACT
Three typically grown Bangladeshi fruit trees, Averrhoa carambola L, Spondias pinnata and
Aegle marmelos possess various therapeutically important properties and their different parts
have been experimented for antioxidant and cytotoxicity activities in various studies.
Antioxidants help prevent free radicals from harming our body with various degenerative
diseases by donating electrons. Antioxidants neutralize the free radicals and reduce the damage
in cells before oxidants can attack. The objective of this study was to evaluate the three fruit
plants leaf extracts and to find their antioxidant content and also In vitro cytotoxic activity using
HeLa cell line. This study may allow us to get a comprehensive analysis on their individual
activities and show susceptible results which will help in further research prospective. As they
are typically grown in Bangladeshi environment and are readily available, they can be screened
to find out which of them contain the highest amount of antioxidant and cytotoxic properties.
Dried powdered leaves of Averrhoa carambola L, Spondias pinnata and A. marmelos were
extracted using a Soxhlet apparatus using, n-hexane, petroleum ether, methanol and chloroform.
The extracts were tested for CUPRAC reducing capacity, total antioxidant capacity, total
phenolic content and total flavonoid content. Cytotoxic analysis was done using HeLa cell line
and cell viability was checked. All three methanolic leaf extracts of the fruits plants showed the
highest CUPRAC reducing capacity when compared against the standard (BHT). For both
Averrhoa carambola L and A. marmelos, petroleum ether extract showed the highest antioxidant
content; 105.8mg/gm, 89.4mg/gm; n-hexane extract showed the highest phenol content;
212.4mg/gm, 686mg/gm and methanol extract showed the highest flavonoid content;
171.6mg/gm, 146mg/gm. For Spondias pinnata, chloroform extract showed the highest total
antioxidant content; 122mg/gm, petroleum ether extract showed the highest phenol content;
280.6mg/gm and methanol extract showed the highest flavonoid content; 189.5mg/gm. In vitro
cytotoxicity test revealed that all extracts of A. marmelos showed <1% viability of HeLa cells,
followed by Averrhoa carambola L extracts, while Spondias pinnata extracts showed the least
cell death. It can be suggested that both Averrhoa carambola L and A. marmelos extracts showed
promising results than Spondias pinnata and they can be further analysed in a more diverse
manner.
Contents

INDEX PAGE NO. I - VII

List of Figures I - IV
List of Tables V - VI
Abbreviations VII
CHAPTER 1 INTRODUCTION PAGE NO. 1 - 18

1. INTRODUCTION 1
1.1 Spondias pinnata (Hogplum/HogApple; Local name Aamra) 2
1.1.1 Taxonomy of the plant 3
1.1.2 Importance of Spondias pinnata 4 - 5
1.2 Averrhoa carambola L. (Star Fruit/Carambola/Kamranga) 6 - 7
1.2.1 Taxonomy of the plant 7
1.2.2 Importance of Averrhoa carambola L. 8 - 9
1.3 Introduction to Aegle marmelos (L.) 10 - 11
1.3.1 Taxonomy of the plant 12
1.3.2 Importance of Aegle marmelos 13 - 18
1.4 Objective of this study 18
Contents

CHAPTER 2 MATERIALS AND METHODS PAGE NO. 20 - 36

2.1 Collection of Spondias pinnata leaves 20
2.1.1 Preparation of Spondias pinnata leaves 20
2.1.2 Extraction of the plant material 20 - 21
2.2 Collection of Averrhoa carambola leaves 21
2.2.1 Preparation of Averrhoa carambola leaves 21
2.2.2 Extraction of the plant material 22
2.3 Collection of Aegle marmelos leaves 22
2.3.1 Preparation of Aegle marmelos leaves 22
2.3.2 Extraction of the plant material 23
2.4 Antioxidant Capacity 23 – 24
2.4.1 Cuprac Reducing Antioxidant Capacity 24 – 26
2.4.2 Total antioxidant capacity 26 – 28
2.4.3 Total phenolic content 28 – 30
2.4.4 Total flavonoid content 31 – 32
2.5 In vitro Cytotoxicity using Animal cell culture 33
2.5.1 Application of animal cell technique 33

2.5.2 Experimental procedure 34
2.5.2.1 Preparation of samples 34
2.5.2.2 Used consumables 34
2.5.2.3 Thawing of cells 34
2.5.2.4 Cell Passage 34
2.5.2.5 Cell counting 35
2.5.2.6 Determination of cytotoxic activity 36

CHAPTER 3 RESULTS PAGE NO. 38 - 71

3.1 Cuprac Antioxidant Capacities 38 – 40
3.2 Total antioxidant Capacites 41 – 46
3.3 Determination of Total Phenolic content 47 – 52
3.4 Determination of Total Flavonoid content 53 – 58
3.5 Determination of Cytotoxicity of the extracts 59 – 71

CHAPTER 4 DISCUSSION PAGE NO. 73- 77

CHAPTER 5 REFERENCES PAGE NO. 79 - 85

APPENDICES PAGE NO. ( i – v)
LIST OF FIGURES

FIGURE FIGURE NAME PAGE NO.
NUMBER
Figure 1 Spondias pinnata fruit 3
Figure 2 Spondias pinnata leaves 3
Figure 3 Averrhoa carambola L. 7
Figure 4 Leaves of A. marmelos 12
Figure 5 A. marmelos Fruit 12
Figure 6 Soxhlet Apparatus 21
Figure 7 Applications of Cell culture technique 33
Figure 8 Hemocytometer 36
Figure 9 Comparative reducing powers of Averrhoa carambola 38
L.extracts and Ascorbic acid & Butylated hydroxyl toluene
(BHT)
Figure 10 Comparative reducing powers of A. marmelos extracts and 39
Ascorbic acid & Butylated hydroxyl toluene (BHT)
Figure 11 Comparative reducing powers of Spondias pinnata extracts 40
and Ascorbic acid & Butylated hydroxyl toluene (BHT)
Figure 12 Calibration curve of ascorbic acid for Averrhoa carambola L 41
Figure 13 Total antioxidant capacities of the different extracts of 42
Averrhoa carambola L. extracts
I
Figure 14 Calibration curve of ascorbic acid for A. marmelos 43
Figure 15 Total antioxidant capacities of the different extracts of A. 44
marmelos extracts
Figure 16 Calibration curve of ascorbic acid for Spondias pinnata 45
extracts
Figure 17 Total antioxidant capacities of the different extracts of 46
Spondias pinnata extract
Figure 18 Calibration curve of Gallic Acid for Averrhoa carambola L. 47
Figure 19 Total phenol contents of the different extracts of Averrhoa 48
carambola L.
Figure 20 Calibration curve of Gallic Acid for A. marmelos 49
Figure 21 Total phenol contents of the different extracts of A. 50
marmelos
Figure 22 Calibration curve of Gallic Acid for Spondias pinnata 51
Figure 23 Total phenol contents of the different extracts of Spondias 52
pinnata
Figure 24 Calibration curve of Quercetin for Averrhoa carambola L. 53
Figure 25 Total flavonoid contents of the different extracts of 54
Averrhoa carambola L.
Figure 26 Calibration curve of Quercetin for A. marmelos 55
Figure 27 Total flavonoid contents of the different extracts of A. 56
marmelos
II