LIVER CANCER TREATMENTS
By Dimas Prasetyo
11405010
BACHELOR’S DEGREE in
FOOD TECHNOLOGY
LIFE SCIENCES AND TECHNOLOGY
SWISS GERMAN UNIVERSITY The Prominence Tower
Jalan Jalur Sutera Barat No. 15, Alam Sutera Tangerang, Banten 15143 - Indonesia
August 2018
Revision after the Thesis Defense on the 25th of July 2018
Dimas Prasetyo STATEMENT BY THE AUTHOR
I hereby declare that this submission is my own work and to the best of my knowledge, it contains no material previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any other degree or diploma at any educational institution, except where due acknowledgement is made in the thesis.
Dimas Prasetyo
_____________________________________________
Student Date
Approved by:
Dr. rer. nat. Maruli Pandjaitan
_____________________________________________
Thesis Advisor Date
Dr. rer. nat. Filiana Santoso
_____________________________________________
Thesis Co-Advisor Date
Dr. Dipl. -Ing. Samuel P. Kusumocahyo
_____________________________________________
Dean Date
Revision after the Thesis Defense on the 25th of July 2018
Dimas Prasetyo ABSTRACT
CYTOTOXICITY OF ANNONA MURICATA EXTRACT WITH ADDITION OF ALLIUM SATIVA, CURCUMA MANGGA, AND BROMELAIN FOR BREAST AND
LIVER CANCER TREATMENTS
By Dimas Prasetyo
Dr. rer. nat. Maruli Pandjaitan, Advisor Dr. rer. nat. Filiana Santoso, Co-Advisor
SWISS GERMAN UNIVERSITY
Soursop leaves, garlic, and turmeric extract were known to have anticancer properties.
A mixture of the 3 extracts (GST) has shown to have an even higher anticancer activity according to past studies. This study aims to utilize bromelain, an enzyme found in pineapple which has shown to be cytotoxic towards cancer cells to further increase the anticancer activity of the GST extract mixture. The analysis was done using NADH oxidase assay, brine shrimp lethality assay and MTT assay.
The analysis of NADH oxidase inhibition rate was done using enzymatic reactions and the addition of bromelain increased the inhibition rate of the GST extract mixture from 41.244% up to 54.378%. The results of the MTT Assay against T47D breast cancer cells showed that the addition of bromelain could increase the anticancer activity of the GST extract mixture by 17.85%, from IC50 value of 32.45 ppm to 27.535 ppm and the activity increases in relation to the amount of bromelain added.
MTT assay was also tested against HepG2 liver cancer cells but showed significantly lower potency even with increased amounts of bromelain. Lastly, brine shrimp lethality assay showed that the addition of bromelain could increase the cytotoxicity of the extracts by 28.38%.
Keywords: Annona muricata, Allium sativa, Curcuma mangga, bromelain, anticancer
Dimas Prasetyo
© Copyright 2018 by Dimas Prasetyo All rights reserved
Dimas Prasetyo DEDICATION
I dedicate this work to everybody who is fighting against cancer and to all the people who have lost the person in their lives to cancer.
Also, to all the people who have supported me throughout my thesis work.
Dimas Prasetyo ACKNOWLEDGEMENTS
Firstly, I would like to thank God who has blessed me with the will and strength to be able to finish this thesis work.
To my family, I would not be able to be who I am today without your everlasting love and support. I cannot thank you enough for everything you have done for me.
I would also like to express profound gratitude to Dr.rer.nat. Maruli Pandjaitan as my advisor and Dr. rer. nat. Filiana Santoso as my co-advisor for their guidance and patience throughout my thesis work.
My heartfelt thanks to Rebecca Wirawan for her endless love, support, and encouragement throughout my thesis work. Thank you for always being there for me and for making me a better person.
I am particularly grateful for the Life Sciences and Technology lecturers who has been such great help during my laboratory work. I would like to thank Mrs. Maria D.P.T. Gunawan Puteri, S.T.P., M.Sc., Ph.D, Ms. Purwanty Rara Azura, Mr. Gerald Justin, S.T, B.Eng and Mr. M. Roziq for their guidance.
I would also like to express great appreciation to Jevon, Latu, William, Jennifer, Amanda, Vimothy and Jansen who have helped me with all my work and non-work related problems.
I would like to offer my special thanks to Tuginah, my maid. She has been such great help to me and my family for the past 22 years and to Ms. Elbakyan and her team, who has helped to remove all barriers in the way of science for students around the world.
Lastly, I thank all the Life Sciences and Technology students. It would not be an understatement to say that these past 4 years would not be worthwhile without your presence.
Dimas Prasetyo TABLE OF CONTENTS
Page
STATEMENT BY THE AUTHOR ... 2
ABSTRACT ... 3
DEDICATION ... 5
ACKNOWLEDGEMENTS ... 6
TABLE OF CONTENTS ... 7
LIST OF FIGURES ... 10
LIST OF TABLES ... 11
CHAPTER 1 - INTRODUCTION ... 12
1.1 Background ... 12
1.2 Research Problems ... 14
1.3 Research Objectives ... 14
1.4 Significance of Study ... 14
1.5 Research Questions ... 15
1.6 Hypothesis ... 15
1.7 Scope ... 15
CHAPTER 2 - LITERATURE REVIEW ... 16
2.1 Cancer Cells ... 16
2.1.1 Warburg Effect ... 17
2.1.3 NADH Oxidase and Production of Lactic acid / Lactate ... 18
2.1.4 NADH Oxidase Assay ... 19
2.2 Soursop (Annona muricata) ... 19
2.3 Annonaceous Acetogenin (AGEs) ... 21
Dimas Prasetyo
2.5 White Turmeric (Curcuma mangga) ... 22
2.6 Bromelain ... 23
2.7 Protease Activity Assay ... 24
2.9 Brine Shrimp Lethality Assay ... 25
CHAPTER 3 – RESEARCH METHODS ... 26
3.1 Venue and Time ... 26
3.2 Material and Equipment ... 26
3.2.1 Raw Materials ... 26
3.2.2 Chemicals ... 26
3.2.3 Equipment ... 27
3.3 Design of Experiment ... 27
3.4 Experimental Procedure ... 29
3.4.1 Preparation of Samples ... 29
3.4.2 Sample Extraction ... 29
3.5 Preparation of Extract Mixtures ... 30
3.5.1 Preparation of Garlic, Soursop Leaves, Turmeric (GST) Extract Mixture for NADH Oxidase Inhibition Analysis ... 30
3.5.2 Preparation of Bromelain Solution for NADH Oxidase Inhibition Analysis . 30 3.5.3 Preparation of Extract Mixture for Brine Shrimp Lethality Assay ... 30
3.5.3.1 Total Soluble Solids Determination ... 30
3.5.3.2 Preparation of Extract Mixture ... 30
3.6 Analytical Procedure ... 31
3.6.1 NADH Oxidase Inhibition ... 31
3.6.1.1 Preparation of Phosphate Buffer Saline (PBS) solution ... 31
3.6.1.2 Preparation of Reagents ... 31
3.6.1.3 Preparation of Chicken Liver Enzyme ... 31
3.6.1.4 Evaluation of NADH Oxidase Inhibition ... 32
Dimas Prasetyo
3.6.2.1 Preparation of Reagents ... 33
3.6.2.2 Preparation of Tyrosine Standard Curve ... 33
3.6.2.3 Determination of Enzyme Activity of Bromelain Powder ... 34
3.6.3 MTT Assay ... 34
3.6.3.1 Cell Harvesting ... 34
3.6.3.2 Cell Counting ... 34
3.6.3.3 Cell Plating ... 35
3.6.3.4 Sample Preparation ... 35
3.6.3.5 Sample Treatment ... 35
3.6.4 Brine Shrimp Lethality Assay ... 36
3.6.4.1 Hatching the Artemia salina Larvae Eggs... 36
3.6.4.2 Making the parent solution ... 36
3.6.4.4 Preparation of Sample Solutions ... 36
3.6.4.3 Placing the larvae into the sample... 37
3.6.4.4 Calculation of the LC50 Value ... 37
CHAPTER 4 – RESULTS AND DISCUSSIONS ... 38
4.1 Extraction of Soursop (Annona muricata) Leaves ... 38
4.2 Extraction of Garlic (Allium sativa) and Turmeric (Curcuma mangga) ... 39
4.3 Nadh Oxidase Inhibition ... 39
4.4 Determination of Enzyme Activity of Bromelain ... 42
4.5 MTT Assay ... 43
4.5.1 Cytotoxicity Against T47D Breast Cancer Cell ... 43
4.5.2 Cytotoxicity Against HepG2 Liver Cancer Cell ... 46
4.6 Brine Shrimp Lethality Assay ... 48
CHAPTER 5 – CONCLUSIONS AND RECOMMENDATIONS ... 52
5.1 Conclusions ... 52
5.1 Recommendations ... 52
GLOSSARY ... 54
Dimas Prasetyo APPENDICES ... 64 CURRICULUM VITAE ... 75
LIST OF FIGURES
Figures Page
Figure 1. Energy producing pathways in normal cells ... 17 Figure 2. Chemical structure of curcumin ... 23 figure 3. Process flowchart... 28 Figure 4. Percentage inhibition of NADH oxidase rate of GST extract added with
various amounts of bromelain ... 42 Figure 5. Logarithmic graph of % mortality vs. concentration of GST extract ... 49 Figure 6. Logarithmic graph of % mortality vs. concentration of GST extract added
with bromelain ... 51
