cytotoxicity of annona muricata extract with addition

TREATMENT

By Kevin Jonathan

14312045

BACHELOR’S DEGREE in

ELECTRICAL ENGINEERING – BIOMEDICAL ENGINEERING CONCENTRATION

FACULTY OF LIFE SCIENCES AND TECHNOLOGY

SWISS GERMAN UNIVERSITY EduTown BSD City

Tangerang 15339 Indonesia

August 2016

Revision after the Thesis Defense on 21

^st

July 2016

Kevin Jonathan

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.

Kevin Jonathan

_____________________________________________

Student Date

Approved by:

Dr. rer. nat Maruli Pandjaitan

_____________________________________________

Thesis Advisor Date

Kholis Abdurachim Audah, M.Sc, Ph.D

_____________________________________________

Thesis Co-Advisor Date

Dr. Dipl.–Ing. Samuel P. Kusumocahyo

_____________________________________________

Dean Date

Kevin Jonathan

ABSTRACT

CYTOTOXICITY OF ANNONA MURICATA EXTRACT WITH ADDITION OF ALLIUM SATIVA AND CURCUMA MANGGA FOR CANCER TREATMENT

By Kevin Jonathan

Dr.rer.nat Maruli Pandjaitan, Advisor

Kholis Abdurachim Audah, M.Sc, Ph.D, Co-Advisor

SWISS GERMAN UNIVERSITY

Soursop leaves (Annona muricata) are composed of a lot of alkaloid chemical constituents such as Annonaceous acetogenins, which are believed to have cytotoxic properties against tumor cell lines. Garlic (Allium sativa) and white turmeric (Curcuma mangga), are also believed to have those properties of inhibiting the proliferation of cancer cells, besides being used as a natural flavor enhancer. The general objective of this research was to prove that the extract of Soursop leaves combined with Garlic and White turmeric is effective to inhibit the growth of cancer cell lines. They will be extracted using ethanol, and later will be distilled to remove all the alcohol remained in the extract, because it was meant to be consumed. MTT assay was done in order to obtain the inhibition of cancer cell activity of the extract. NADH oxidation enzyme inhibition was performed to obtain the optmal concentration, and BSLT was conducted to show the bioactivity of the extract. The extract showed an inhibition result of 59.78% while the LC

₅₀

obtain from the extract using BSLT was 93.73 ppm.

Keywords: Soursop leaves, NADH inhibition activity, Allium sativa, Curcuma

mangga, anti-cancer activity, MTT, Lethal Concentration.

Kevin Jonathan

© Copyright 2016

by Kevin Jonathan

All rights reserved

Kevin Jonathan

DEDICATION

I dedicate this thesis work for my beloved family, and those who supports me all the

time during the thesis work.

Kevin Jonathan

ACKNOWLEDGEMENTS

First of all I would like to express my gratitude to God, for His mercy and grace guided me to finish the thesis work and report.

This research could not have been possible without the help of many people, and thus I would also express my gratitude for my parents and brother who always supported me. And moreover, I would like thank those who assisted and helped me during the research:

 Mr. Dr.rer.nat. Maruli Pandjaitan, as my thesis advisor, for his ideas, help and guidance during thesis work.

 Kholis Abdurachim Audah, M.Sc, Ph.D as my thesis co-advisor, for his support and advices.

 Dr. Muhammad Nursid and staffs in LITBANG Perikanan, for their advices and support.

 Arthur Jonathan, Boby Hartanto, Daniel Agustian, Riswandi Wansyah, and Saineris Surya who always support me and encourage me during my thesis work.

And lastly for all SGU staffs, officers, and all colleagues, and for all those who have helped me completing my thesis work. I hope that the result of my research could be useful.

Tangerang, June 17

^th

, 2016

Kevin Jonathan

Kevin Jonathan

TABLE OF CONTENTS

STATEMENT BY THE AUTHOR ... 2

ABSTRACT ... 3

DEDICATION... 5

ACKNOWLEDGEMENTS ... 6

TABLE OF CONTENTS ... 7

LIST OF FIGURES ... 9

LIST OF TABLES ... 10

CHAPTER 1 - INTRODUCTION ... 11

1.1 Background ... 11

1.2 Research Problems ... 12

1.3 Research Objectives ... 12

1.4 Significance of Study ... 12

1.5 Research Questions ... 13

1.6 Hypothesis ... 13

CHAPTER 2 - LITERATURE REVIEW ... 14

2.1 Annona muricata (Soursop) ... 14

2.2 Annonaceous acetogenins ... 14

2.3 Allium sativa (Garlic) ... 16

2.4 Curcuma mangga (White Turmeric) ... 18

2.5 Microculture tetrazolium assay ( MTT Assay) ... 19

2.6 Spectrophotometry ... 20

2.7 Rotary Evaporator ... 21

CHAPTER 3 - RESEARCH METHODS ... 22

3.1 Venue and Time ... 22

3.2 Material and Equipments ... 22

3.2.1 Raw Material ... 22

3.2.2 Chemical ... 22

3.2.3 Equipments ... 22

3.3 Research Procedure... 23

3.3.1 Design of Experiment ... 23

3.3.2 Experiment Procedure ... 23

3.4 Observations ... 26

3.5 Analytical Procedure ... 26

Kevin Jonathan

3.5.1 NADH Oxidation Inhibition ... 26

3.5.1.1 Preparation of Phosphate Buffer Saline (PBS) solution ... 26

3.5.1.2 Preparation of Chicken Liver Serum ... 26

3.5.1.3 NADH oxidase inhibition evaluation ... 26

3.5.2 Brine Shrimp Lethality Test (BSLT) ... 27

3.5.2.1 Hatching of Artemia saline Leach ... 27

3.5.2.2 Preparation of Sample ... 27

3.5.3 MTT Assay ... 28

3.5.3.1 Cell Harvesting ... 28

3.5.3.2 Cell counting... 29

3.5.3.3 Plating ... 29

3.5.3.4 Sample Preparation ... 29

3.5.3.5 Sample treatment ... 29

3.5.3.6 Addition of MTT reagent ... 29

3.5.3.7 Data Analysis ... 30

CHAPTER 4 – RESULTS AND DISCUSSIONS ... 31

4.1 Extraction of Annoceous Acetogenins ... 31

4.2 Extraction of Allium sativa and Curcuma mangga ... 32

4.3 Production Process of Soursop Leaves Extract ... 32

4.4 Inhibition Activity of Extract... 32

4.4 Determination of Concentration of the Extract ... 34

4.5 LC50 from Brine Shrimp Lethality Test ... 35

4.6 Microculture Tetrazolium Assay ( MTT Assay) ... 38

CHAPTER 5 – CONCLUSIONS AND RECOMMENDATIONS ... 40

5.1 Conclusions ... 40

5.2 Recommendation ... 40

REFERENCES... 41

APPENDICES ... 44

Curriculum Vitae ... 47