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THE EFFECT OF Annona muricata TO INCREASE CXCL10 IN THE BRAIN STUDY IN CEREBRAL MALARIA OF SWISS MICE
PENGARUH ANNONA MURICATA UNTUK MENINGKATKAN CXCL10 DI OTAK STUDI PADA SEREBRAL MALARIA SWISS ALBINO MICE
Thesis
For requirements master degrees
Master of Biomedical Sciences
SUMIA M. ALI MATUG 22010114419008
FACULTY OF MEDICINE
DIPONEGORO UNIVERSITY SEMARAING 2016
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DECLARATION
I hereby declare that this thesis is my own work and has not been submitted in any from for another degree or diploma at any university or other institution of tertiary education, there are no elements belonging plagiarism forth in Decree No 17 of 2010. Information derived from the published or unpublished work of others has been acknowledged in the text and a list of reference is given.
Semarang, June, 2016
SUMIA M. ALI MATUG
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CURRICULUM VITAE
A. Identity
Name : Sumia M. Ali Matug
Sex : Female
Place and Date of Birth : Mesallata / October, 17th 1981
Religion : Muslim
Address : Mesallata, Libya
B. Educational Background No Name of
School/Institution
Location Degree Year
1 Aleahd Aljadid Mesallata, Libya Primary 1996
2 Aleahd Aljadid Mesallata, Libya Secondary 2000
3 Mesallata University Mesallata, Libya Bachelor 2007
4 Diponegoro University Semarang, Indonesia Master 2016
C. Job Experience
1. 2007 – 2013: Teacher for Laboratory Medical Technology
D. Family History
1. Name of father : Mansur Ali Matug 2. Name of mother : SaAla Muhammad
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FOREWORD
Assalamu’alaikum Wr. Wb.
Praise to Allah Almighty for all grace and guidance that thesis with the title “The Effect of Annona muricata to Increase CXCL10 in The Brain Study in Cerebral Malaria of Swiss Mice” can be resolved. This thesis is structured to meet one of the requirements to obtain a Master degree in Biomedical Sciences (MSi. Med) in the field of Immunology at the Faculty of Medicine, University of Diponegoro.
I realized that without the help and guidance of the various parties, it is not easy for me to finish this thesis. Therefore, on this occasion, the author would like to express respect and gratitude as possible to:
1. Prof. Dr. H. Yos Johan Utama, S.H., M.Hum. Rector of Diponegoro University who has provided an opportunity for authors to improve science learning.
2. Prof. Dr. dr. Tri Nur Kristima DMM., M.Kes. Dean of the Faculty of Medicine, University of Diponegoro who has provided the opportunity for the author to participate in education.
3. Dr. dr. RA. Kisdjamiatun RMD, M.Sc. Head Master of Biomedical Sciences Faculty of Medicine, University of Diponegoro who has provided motivation and support to the author to complete his education on time.
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4. Dr. dr. Awal Prasetyo, M.Kes., Sp. THT-KL as the First Counselor who has been willing to invest time, energy and mind to guide the writer for completing this thesis.
5. Dr. dr. RA. Kisdjamiatun RMD, M.Sc. as the Second Counselor who has been willing to take the time, effort, and thought to guide the writer for completing this thesis.
6. dr. Sudaryanto, M.Pd.Ked, as the Head of the Laboratory of Parasitology, Faculty of Medicine, University of Diponegoro and the entire laboratory staff who have given permission Parasitology and support for research.
7. Prof. Sultana M. Faradz, PhD. and the entire staff Laboratory Cebior who have given permission and support for research.
8. Professors and Lecturers in the Master of Biomedical Sciences Faculty of Medicine, University of Diponegoro who has given his knowledge during his education writer.
9. All employees / academic staff in the program as Master of Biomedical Sciences Faculty of Medicine, University of Diponegoro who have provided assistance for the author was educated.
10. Parents and the whole loved ones who have given prayers and support during the authors studied.
11. Fellow students of Master of Biomedical Sciences Faculty of Medicine, University of Diponegoro 2014 which has been jointly studying and providing support to authors.
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12. To all those who have a lot to provide assistance, but can not mention one by one author.
With limited knowledge, the authors expect criticism and constructive suggestions for the perfection of this thesis.
Finally, the authors hope that Allah is pleased to reply to all the good of all those who have helped this thesis. Hopefully, this thesis can be useful for the development of science, especially Biomedicscience.
Wassalamualaikum Wr. Wb.
Semarang, June, 2016
SUMIA M. ALI MATUG
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TABLE OF CONTENTS
Page
Cover ... i
Approval ... ii
Declaration ... iii
Curriculum Vitae ... iv
Foreword ... v
Table of Contents ... viii
List of Abbreviations ... xi
List of Figures ... xiii
List of Table ... xiv
List of Appendixes ... xvi
Abstract ... xvii
Abstrak ...xviii
CHAPTER I INTRODUCTION ... 1
1.1. Background ... 1
1.2. Research Questions ... 4
1.3. Research Objective ... 4
1.3.1. General Objective ... 4
1.3.2. Specific Objective ... 4
1.4. Research Benefits ... 4
1.5. Research Originality ... 5
CHAPTER II LITERATURE REVIEW ... 7
2.1. Malaria ... 7
2.1.1 Life Cycle Plasmodium falciparum ... 7
2.1.2 Pathogenesis of Malaria ... 8
2.2 CXCL9 and CXCL10 in Experimental CM ... 9 2.3 CXCL9 and CXCL10 in Plasmodium berghei
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ANKA Infected ... 12
2.4 Plasmodium berghei ANKA ... 14
2.4.1 Taxonomy Plasmodium berghei ANKA ... 14
2.4.2 P. berghei ANKA Life Cycle ... 15
2.5 Swiss Mouse Brain Infected with P. berghei Anka .... 16
2.6 Astrocyte Function in the Brain ... 18
2.7 Relation Between Astrocyte and Th1 Cell and Mechanism Astrocyte in Regulating Th1 Cell .... 20
2.8 Annona muricata Linn. ... 22
2.8.1 Taxonomy ... 22
2.8.2 Morphology of Annona muricata ... 23
2.8.3 Medicine Use of Annona muricata ... 24
2.9 Theoretical Framework ... 26
2.10 Conceptual Framework ... 27
2.11 Hypothesis ... 27
CHAPTER III RESEARCH METHOD ... 28
3.1 Scope of Study ... 28
3.2 Design of Study ... 28
3.3 Population and Sample ... 29
3.3.1 Population ... 29
3.3.2 Sample ... 30
3.3.3 Sample Size ... 30
3.3.4 Inclusion Criteria ... 31
3.3.5 Drop Out ... 31
3.4 Variable of Study ... 31
3.5 Operational Definitions ... 31
3.6 Data Analysis ... 32
3.7 Research Instrument and Consumables ... 32
3.7.1 Instrument ... 32
3.7.2 Consumable Materials ... 33
3.8 Study Protocol ... 33
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3.9 Research Flow ... 35
CHAPTER IV RESULT AND DISCUSSION ... 36
4.1. Result ... 36
4.1.1. Descriptive of CXCL10 Expression in Swiss Mice Brain ... 36
4.1.2. CXCL10 Expression in the Brain ... 37
4.1.3. Kappa Test ... 37
4.2. Discussion ... 39
CHAPTER V CONCLUSION AND SUGGESTION ... 46
5.1. Conclusion ... 46
5.2. Limitations ofthe Study ... 46
References ... 47
Appendix ... 48
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LIST OF ABBREVIATIONS
APC : antigen presenting cells BBB : blood brain barrier
CM : cerebral malaria
CSF : cerebral spinal fluid CXCR3 : (C-X-C motif) receptor 3 ECM : experimental cerebral malaria GPI : glikosilfosfatidilinasitol HRP-1 : histidine-rich protein-1
ICAM-1 : Intercellular adhesion molecule-1 IFN-γ : interferon-gamma
IL-1 : interleukin-1
MMP : Matrix metalloproteinases NK : natural killer
NKT : natural killer T NO : nitric oxide
PbA : P. berghei ANKA
pRBC : parasitized red blood cell RES : reticulo endothelial system RPE : retinal pigment epithelial TNF-α : tumor necrosis factor-alpha
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LIST OF FIGURES
Figure 1. Life Cycle Plasmodium ... 8
Figure 2. Annona muricata Linn. ... 23
Figure 3. Theoretical Framework ... 26
Figure 4. Conceptual Framework ... 27
Figure 5. Design of Study ... 28
Figure 6. Research Flow ... 35
Figure 7. Graph Box plot of CXCL10 expressed in the healthy or PbA-inoculated swiss mice with or without A. muricata treatment ... 36
Figure 8. Picture of CXCL10 ... 37
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LIST OF TABLES
Table 1. Previous report related to study ... 5 Table 2. Operational Definition ... 31 Table 4.1. Kappa test for examination CXCL10 level ... 38 Table 4.2. Statistical analysis of CXCL10 expressed in the brain
of healthy or PbA-inoculated mice with or without
A.muricata treatment ... 38
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LIST OF APPENDIX
Appendix 1. Preparation Extracts of Annona muricata Leaves ... 60
Appendix 2. Index Calculation Parasitemia Mice Donor ... 61
Appendix 3. Making the Inoculums From Donor Mice ... 63
Appendix 4. Weight Data of Swiss Mice ... 64
Appendix 5. Result Analysis of CXCL10 IHC ... 66
Appendix 6. Statistical Analysis ... 67
Appendix 7. Ethical Clearance ... 80
Appendix 8. Certificate of Analysis Annonae muricatae Folium ... 81
Appendix 9. Documentation of Research ... 82
xiv ABSTRACT
THE EFFECT OF ANNONA MURICATA TO INCREASE CXCL10 IN THE BRAIN STUDY IN CEREBRAL MALARIA OF SWISS MICE
Sumia M. Ali Matug
BACKGROUND: Malaria is a common and life-threatening disease in many tropical and subtropical areas. A study of cerebral malaria (CM) in children demonstrated that out of 36 biomarkers, only CXCL10 was serum marker independently associated with CM mortality. Objective of this study is to prove that A. muricata may decrease the CXCL10 in Plasmodium berghei ANKA (PbA) inoculated swiss albino mice.
METHOD: Study design was experimental study, by used Randomized Post test Only Control Group Design. Thirthy six swiss mices which divided into 6 groups.
C(-) is healthy mice, C(+) is group with inoculated PbA, X1 (non inoculated PbA and A. muricata dosage 100 mg/kg BW), X2 (non inoculated PbA and A. muricata dosage 150 mg/kg BW), X3 (inoculated PbA and A. muricata dosage 100 mg/kg BW), X4 (inoculated PbA and A. muricata dosage 150 mg/kg BW).
RESULT: Significant difference was found among the studied groups (p=0.003).
Positive control group was signiticantly expressed lower CXCL10 than those of healthy groups without or with A. muricata treatment (negative control, p=0.008;
X1, p=0.045; or P2, p=0.012). X3 group showed CXCL10 expression comparable with those of healthy mice (negative control, X1 and X2), meanwhile the expression was significantly higher than positive control and X4 groups (p=0.012 and p=0.028, respectively). X4 group showed CXCL10 expression significantly lower than healthy groups which were negative control and P2 groups (p=0.011 and p=0.016, respectively).
CONCLUSION: Annona muricata can’t decrease CXCL10 in PbA inoculated swiss albino mice.
Keywords: Annona muricata, P. berghei ANKA, CXCL10.
xv ABSTRAK
THE EFFECT OF ANNONA MURICATA TO INCREASE CXCL10 IN THE BRAIN STUDY IN CEREBRAL MALARIA OF SWISS MICE
PENGARUH ANNONA MURICATA UNTUK MENINGKATKAN CXCL10 DI OTAK STUDI PADA SEREBRAL MALARIA SWISS ALBINO MICE
Sumia M. Ali Matug
LATAR BELAKANG: Malaria adalah penyakit yang umum dan mengancam jiwa di banyak daerah tropis dan subtropis. Sebuah studi dari cerebral malaria pada anak-anak menunjukkan bahwa dari 36 biomarker, hanya CXCL10 adalah penanda serum independen terkait dengan kematian CM. Penting untuk meneliti tentang CXCL10. Tujuan dari penelitian ini adalah untuk mengetahui A. muricata dapat menurunkan CXCL10 pada tikus swiss albino yang diinokulasi oleh Plasmodium berghei ANKA(PbA).
METODE: Rancangan penelitian adalah penelitian eksperimental, dengan menggunakan Acak Posting uji Hanya Control Group Design. Tiga puluh enam tikus swiss albino yang terbagi menjadi 6 kelompok. C(-) adalah tikus yang sehat, C(+) adalah grup dengan diinokulasi PbA, X1 (diinokulasi PbA dan ekstrak A.
muricata 100 mg / kg BB), X2 (tidak diinokulasi PbA dan ekstrak A. muricata 150 mg / kg BB), X3 (diinokulasi PbA dan ekstrak A. muricata 100 mg / kg BB), X4
(diinokulasi PbA dan ekstrak A. muricata 150 mg / kg BB).
HASIL: Perbedaan yang signifikan ditemukan antara kelompok dipelajari (p = 0,003). Kelompok kontrol positif signiticantly CXCL10 lebih rendah dibandingkan kelompok sehat tanpa atau dengan pengobatan A. muricata (kontrol negatif, p = 0,008; X1, p = 0,045; atau X2, p = 0,012). Kelompok X3 menunjukkan ekspresi CXCL10 sebanding dengan tikus-tikus yang sehat (kontrol negatif, X1
dan X2), sementara ekspresi secara signifikan lebih tinggi dari kontrol dan X4
kelompok positif (p = 0,012 dan p = 0,028, masing-masing). Kelompok X4
menunjukkan ekspresi CXCL10 signifikan lebih rendah daripada kelompok yang sehat yang kontrol dan X2 negatif kelompok (p = 0,011 dan p = 0,016, masing- masing).
KESIMPULAN: Annona muricata tidak dapat menurunkan CXCL10 pada tikus swiss albino yang diinokulasi oleh PbA.
Kata kunci: Annona muricata, P. berghei ANKA, CXCL10.
