KARYA TULIS AKHIR

PENGARUH EKSTRAK BIJI COKLAT (Theobroma cacao) SEBAGAI

ANTIOKSIDAN TERHADAP REGRESI PLAK ATEROMA DINDING

ARKUS AORTA TIKUS PUTIH JANTAN (Rattus norvegicus strain wistar)

ATEROSKLEROTIK

Oleh:

SIDIKA YUNIA MUYASYARAHMA

201110330311017

UNIVERSITAS MUHAMMADIYAH MALANG

FAKULTAS KEDOKTERAN

2015

i

LEMBAR PENGESAHAN

LAPORAN HASIL PENELITIAN

Telah disetujui sebagai hasil penelitian

untuk memenuhi persyaratan

Pendidikan Sarjana Fakultas Kedokteran

Universitas Muhammadiyah Malang

Tanggal 23 Januari 2015

Pembimbing I

Prof. Dr. dr. H. Djoni Djunaedi, Sp.PD., KPTI., FINASIM

Pembimbing II

dr. Yuliono Trika Nur Hasan, Sp.M

Mengetahui,

Dekan Fakultas Kedokteran

Universitas Muhammadiyah Malang

dr. Irma Suswati M.Kes.

ii

dr. Kusuma Andriana, Sp.OG Anggota

iii

KATA PENGANTAR

Segala puji bagi Allah SWT atas segala rahmat dan hidayah-Nya, shalawat

serta salam terlimpahkan kepada Nabi Muhammad SAW, keluarga dan para

sahabatnya. Syukur alhamdulillah, penulis telah berhasil menyelesaikan karya

tulis akhir yang berjudul “PENGARUH EKSTRAK BIJI COKLAT

(Theobroma cacao) SEBAGAI ANTIOKSIDAN TERHADAP REGRESI

PLAK ATEROMA DINDING ARKUS AORTA TIKUS PUTIH JANTAN

(Rattus norvegicus strain wistar) ATEROSKLEROTIK”.

Dalam penyelesaian karya tulis akhir ini penulis ingin mengucapkan

terima kasih yang tak terhingga kepada :

1. dr. Irma Suswati, M.Kes selaku Dekan Fakultas Kedokteran Universitas

Muhammadiyah Malang.

2. dr. Moch. Ma’roef, Sp.OG selaku Pembantu Dekan I Fakultas Kedokteran

UMM serta pembimbing I dalam karya tulis akhir ini.

3. dr. Rahayu, Sp.S selaku Pembantu Dekan II Fakultas Kedokteran UMM.

4. dr. Iwan Sys Indrawanto, Sp.KJ selaku Pembantu Dekan III Fakultas

Kedokteran UMM.

5. Prof. Dr. dr. H. Djoni Djunaedi, Sp.PD., KPTI., FINASIM selaku

pembimbing 1 atas bimbingan, dukungan dan saran yang telah diberikan

dalam penyusunan karya tulis akhir ini.

6. dr. Yuliono Trika Nur Hasan, Sp.M selaku pembimbing 2 atas bimbingan,

dukungan dan saran yang telah diberikan dalam penyusunan karya tulis akhir

ini.

iv

7. dr. Kusuma Andriana, Sp. OG selaku penguji atas bimbingan, dukungan dan

saran yang telah diberikan dalam penyusunan karya tulis akhir ini.

8. dr. Soebarkah Basuki, Sp.PA selaku pembimbing patologi anatomi atas

bimbingan, dukungan dan saran yang telah diberikan dalam penyusunan

karya tulis akhir ini.

9. Mbak Patmawati, Mas Nyono, Mas Miftah, Pak Joko dan segenap Asisten

Lab. Patologi Klinik Universitas Muhammadiyah Malang atas bimbingan,

dukungan, dan saran yang telah diberikan dalam penelitian.

10. Ayahanda Drs H. Nur Rahman, MM, Ibunda Hj. Puji Rahayu, S.Pd, serta

dr.Devy Yuspita Rahma dan Dinsa Okta Rahmawati yang selalu memberikan

kasih sayang, do’a, dukungan, semangat, kepercayaan dan segalanya demi

keberhasilan penulis.

11. Semua teman-teman (Firman, Ro’di, Galang, Wahyu, Priangga, Yusuf,

Brando, Fahmi, Faris, Aulia, Adhinda, Prastiti, Nada, Adhe, Pamela, dan

Dendy) yang telah memberikan bantuan dan dorongan baik moril maupun

materil sehingga terselesaikannya penyusunan tugas akhir ini dengan baik.

Karya tulis akhir ini masih jauh dari kesempurnaan, penulis mohon maaf

yang sebesar-besarnya dan mengharapkan saran dan kritik yang membangun.

Semoga karya tulis ini dapat menambah wawasan dan bermanfaat bagi semua

pihak.

Malang, Januari 2015

Penulis

v

ABSTRAK

Muyasyarahma, Sidika Yunia. 2014. Pengaruh Ekstrak Biji Coklat (Theobroma

cacao) sebagai Antioksidan Terhadap Regresi Plak Ateroma Dinding Arkus

Aorta Tikus Putih Jantan (Rattus norvegicus strain wistar) Aterosklerotik.

Tugas akhir. Fakultas Kedokteran Universitas Muhammadiyah Malang.

Pembimbing: (1) Djoni Djunaedi* (2) Yuliono Trika Nur Hasan**

Latar Belakang: Penyakit jantung koroner (PJK) telah menjadi penyebab utama

kematian di dunia. Penyebab utama PJK adalah penyempitan arteri koronaria

besar di bagian proksimal akibat aterosklerosis. Pada aterosklerosis terjadinya

disfungsi endotel akibat terjadi penurunan fraksi antioksidan yang dapat

menstimulasi produksi Nitric Oxide (NO) yang berfungsi menghambat adhesi dari

monosit dan platelet, kemotaksis leukosit, dan oksidasi LDL. Tujuan: Membuktikan pengaruh pemberian ekstrak coklat sebagai antioksidan

terhadap regresi plak ateroma dinding arkus aorta tikus putih jantan (Rattus

norvegicus strain wistar) aterosklerotik

Metode: Eksperimental dengan menggunakan rancangan The Post Test Only

Control Group Design. Penelitian ini menggunakan 5 kelompok perlakuan yang

masing-masing terdiri dari 5 tikus (kontrol negatif, kontrol positif, ekstrak biji

coklat dosis 1,8mg/200gBB/hari, 3,6mg/200gBB/hari dan 5,4mg/200gBB/hari).

Preparat kemudian dilihat dan dianalisis ketebalan tunika intima dan media arkus

aortanya.

Hasil Penelitian: Uji Tukey 1% menujukkan bahwa pemberian dosis

3,6mg/200gBB/hari mempunyai efek yang sama dengan dosis

5,4mg/200gBB/hari. Pemberian ekstrak biji coklat berpengaruh sangat kuat secara

negatif terhadap ketebalan dinding aorta (R= -0,862). Analisis regresi linier

menunjuk dosis 6,28mg/200gBB/hari tikus sebagai dosis optimal pada penelitian

ini.

Kesimpulan: Ekstrak biji coklat (Theobroma cacao) terbukti mempunyai

aktivitas antioksidan yang dapat meregresi plak ateroma dinding arkus aorta tikus

putih jantan aterosklerotik.

Kata Kunci: Biji Coklat, Flavonoid, Nitric Oxide, Aterosklerosis.

* : Guru Besar Ilmu Penyakit Dalam konsultan Penyakit Tropis dan Infeksi, FK UMM

** : Staff pengajar Ilmu Penyakit Mata, FK UMM

vi

ABSTRACT

Muyasyarahma, Sidika Yunia. 2014. The Effect of Cocoa Extract (Theobroma

cacao) as An Antioxidant To Regress The Atheroma Plaque of Aortic Arc

Wall In Atherosclerotic Male White Rats (Rattus norvegicus strain wistar).

Final project, Medical Faculty of Muhammadiyah Malang University.

Advisors: (1) Djoni Djunaedi* (2) Yuliono Trika Nur Hasan **

Background: Coronary Heart Disease (CHD) has been proven as the main cause

of death in the world. The major cause of CHD is the narrowing of proximal

coronary artery caused by atherosclerosis may due to the decreasing of antioxidant

fraction that has been identified in cacao beans which stimulates the production of

Nitric Oxide (NO) which posses an ability to prevent leukocytes and platelet

adhesion, leukocyte chemotactic and Low Density Lipoprotein (LDL) oxidation.

Purpose: Proving the effect of cocoa extract as an antioxidant to regress the

atheroma plaque of aortic arc wall in atherosclerotic male white rats.

Method: The study was experimental with The Post Test Only Control Group

Design which consisted of 5 groups, each group consisted of 5 rat, (negative

control, positive control, and cocoa extract 1,8mg/200gBW/day,

3,6mg/200gBW/day and 5,4mg/200gBW/day). Tunica intima and media thickness

of the blood vessels were observed and analyzed.

Result: Tukey 1% test showed that dose 3,6mg/200gBB/day had the same effect

as dose 5,4mg/200gBB/day. Cocoa extract had very strong negative effect on the

arcus aortic wall thickness (R= -0,862). The linear regression analysis pointed to a

dose of 6,28mg/200gBB/day as the optimal dose in this study.

Conclusion: Cocoa extract approved have a potential antioxidant activity to

regress the atheroma plaque of aortic arc wall in atherosclerotic male white rats.

Key words: Cacao beans, Flavonoid, Nitric Oxide, Atherosclerosis

* : Professor of The Internal Medicine Department, Tropico-Infectiologist Consultant,

FK UMM

** : Lecturer of The Eye Medicine Department, FK UMM

vii

DAFTAR ISI

LEMBAR PENGESAHAN………………………………………………. i

LEMBAR PENGUJIAN………………………………………………….. ii

KATA PENGANTAR…………………………………………………….. iii

ABSTRAK……………………………………………………………….... v

ABSTRACT……………………………………………………………….... vi

DAFTAR ISI………………………………………………………………. vii

DAFTAR SINGKATAN.............................................................................. x

DAFTAR GAMBAR.................................................................................... xi

DAFTAR TABEL ........................................................................................ xii

BAB 1 PENDAHULUAN........................................................................... 1

1.1 Latar Belakang......................................................................................... 1

1.2 Rumusan Masalah.................................................................................... 3

1.3 Tujuan Penelitian..................................................................................... 3

1.3.1 Tujuan Umum................................................................................ 3

1.3.2 Tujuan Khusus............................................................................... 4

1.4 Manfaat Penelitian................................................................................... 4

1.4.1 Manfaat Akademik........................................................................ 4

1.4.2 Manfaat Klinis............................................................................... 4

BAB 2 TINJAUAN PUSTAKA..................................................................... 5

2.1 Aterosklerosis ............................................................................................ 5

2.1.1 Definisi ………………………………………………………..... 5

2.1.2 Etiologi ………………………………………………………… 6

2.1.3 Faktor Resiko …………………………………………………... 6

2.1.4 Klasifikasi Lesi Aterosklerosis ………………………………… 10

2.1.5 Sel-Sel yang Terlibat dalam Aterosklerosis ………………….... 12

2.1.6 Patogenesis Aterosklerosis …………………………………….. 14

viii

2.2 Nitrite Oxide (NO) .................................................................................. 17

2.2.1 Produksi NO ............................................................................... 18

2.2.2 Fungsi NO .................................................................................. 18

2.3 Reactive Oxygen Species (ROS) .............................................................. 19

2.4 Coklat (Theobroma cacao) ...................................................................... 21

2.4.1 Taksonomi .................................................................................. 21

2.4.2 Morfologi .................................................................................... 21

2.4.3 Biji Theobroma cacao ................................................................ 23

2.4.4 Manfaat Biji Theobroma cacao ................................................. 24

2.5 Peran Flavonoid Biji Theobroma cacao sebagai Antioksidan ................ 25

2.5.1 Flavanol ...................................................................................... 26

2.5.2 Procyanidin ................................................................................. 27

2.6 Bioavaibilitas Biji Coklat (Theobroma cacao) ........................................ 29

BAB 3 KERANGKA KONSEP DAN HIPOTESIS .................................... 30

3.1 Kerangka Konsep ...................................................................................... 30

3.2 Hipotesis ................................................................................................... 32

BAB 4 METODE PENELITIAN .................................................................. 33

4.1 Rancangan Penelitian ................................................................................ 33

4.2 Waktu dan Tempat Pelaksanaan Penelitian .............................................. 33

4.3 Populasi dan Sampel Penelitian ................................................................ 33

4.3.1 Populasi ......................................................................................... 33

4.3.2 Sampel ........................................................................................... 33

4.3.3 Teknik Sampling ........................................................................... 33

4.3.4 Estimasi Besar Sampel .................................................................. 34

4.4 Karakteristik sampel penelitin .................................................................. 35

4.5 Variabel dan Definisi Operasional ............................................................ 35

4.5.1 Variabel ........................................................................................ 35

4.5.2 Definisi Operasional ..................................................................... 35

4.6 Dasar penentuan dosis Ekstrak Biji Coklat .............................................. 36

4.7 Bahan dan Instrumen penelitian ................................................................ 37

4.7.1 Bahan ............................................................................................. 37

ix

4.7.2 Instrumen ....................................................................................... 38

4.8 Alur penelitian .......................................................................................... 40

4.9 Prosedur penelitian .................................................................................... 41

4.9.1 Pembagian Kelompok Tikus ......................................................... 41

4.9.2 Adaptasi ......................................................................................... 41

4.9.3 Pemberian Diet Aterogenik ........................................................... 41

4.9.4 Pemberian Ekstrak Biji Coklat ..................................................... 42

4.9.5 Proses Anastesi Dan Pembedahan Hewan Coba ........................... 42

4.9.6 Pembuatan Sediaan Histopatologi ................................................. 43

4.9.7 Analisa Data .................................................................................. 44

BAB 5 HASIL PENELITIAN DAN ANALISIS DATA.............................. 47

5.1 Gambaran Mikroskopis Dinding Arkus Aorta.............................. 47

5.2 Analisis Data ............................................................................... 49

BAB 6 PEMBAHASAN............................................................................ ..... 54

BAB 7 KESIMPULAN DAN SARAN .......................................................... 59

7.1 Kesimpulan ................................................................................... 59

7.2 Saran .............................................................................. ............... 59

DAFTAR PUSTAKA

LAMPIRAN

x

DAFTAR SINGKATAN

Apo (a) : Apolipoprotein (a)

BH4 : Tetrahydrobiopterin

CAD : Coronary Artery Disease

EDRF : Endothelium Derived Relaxing Factor

EGF : Epidermal Growth Factor

ET : Endothelin-1

FGF : Fibroblast Growth Factor

HDL : High Density Lipoprotein

HoCys : Homocysteins

LDL : Low Density Lipoprotein

LDL-C : Low Density Lipoprotein-Cholesterol

M-CSF : Macrophage Colony Stimulating Factor

NO : Nitric Oxide

eNOS : endothelial Nitric Oxide Synthase

Ox-LDL : Oxidized LDL

PDGF : Platelet Derived Growth Factor

PGE2 : Prostaglandin E2

PGG2 : Prostaglandin G2

PGH2 : Prostaglandin H2

PGI2 : Prostaglandin I2

ROS : Reactive Oxygen Species

s-ICAM : soluble- Intracellular Adhesion Molecule

s-VCAM : soluble- Vascular Cellular Adhesion Molecule

TGF-β : Transforming Growth Factor-β

xi

DAFTAR GAMBAR

Gambar 2.1 Pembuluh Darah Aterosklerotik ........................................... 5

Gambar 2.2 Patofisiologi Aterosklerosis ................................................. 16

Gambar 2.3 Theobroma cacao ................................................................ 21

Gambar 2.4 Biji Theobroma cacao ......................................................... 23

Gambar 2.5 Komponen Flavonoid .......................................................... 25

Gambar 2.6 Struktur Kimia Flavanol ...................................................... 26

Gambar 2.7 Struktur procyanidin ............................................................ 27

Gambar 2.8 Mekanisme Antioksidan Biji Coklat ................................... 29

Gambar 5.1 Gambaran Mikroskopis Ketebalan Dinding Arkus Aorta

Tiap Perlakuan (Perbesaran 400x) ………………………… 47

Gambar 5.2 Kurva Hubungan Antar Dosis Ekstrak Biji Coklat dengan

Ketebalan Plak Tunika Intima dan Media Dinding Arkus

Aorta ……………………………………….……………... 51`

xii

DAFTAR TABEL

Tabel 4.1 Konversi Dosis Antar Spesies ..................................................... 36

Tabel 5.1 Pengaruh Ekstrak Biji Coklat terhadap Ketebalan Plak Tunika

Intima dan Media Dinding Arkus Aorta Setelah Perlakuan…… 48

Tabel 5.2 Perbandingan Dosis Ekstrak Biji Coklat terhadap Ketebalan Plak

Tunika Intima dan Media Dinding Arkus Aorta Setelah

Perlakuan …………………………………………………….... 50

xiii

DAFTAR PUSTAKA

Afoakwa, E. O., Paterson, A., Fowler, M et al. 2008. Flavor Formation and

Character in Cocoa and Chocolate: A Critical Review. Critical Reviews in

Food Science and Nutrition, 48 (9): 840 – 857.

Andújar, I., Recio, M.C., Giner, R.M. et al. 2012. Cocoa polyphenols and their

potential benefits for human health. Oxidative Medicine and Cellular

Longevity, art. no. 906252, 23 p.

ATP III (Adult Treatment Panel III). The third report of the NCEP Expert Pannel

Executive Summary. 2001. Detection Evaluation and Treatment of The

High Blood Cholesterol in Adult, NCEP, NHL and Blood Institute, NH.

NIH Publication No. 01-3679, May 2001.

Bagchi, Stohs, Das et al. 2000. Free radicals and grape seed proanthocyanidin

extract: importance in human health and disease prevention. Toxicology.

148 2000), 187-197

Blanco-Vaca, F., Escola-Gil, J.C., Martin Campos, J.M. et al. 2001. Role of apoA-

II in lipid metabolism and aterosklerosis:advances in the study of an

enigmatic protein. J Lipid Res. 42: 1727-1739.

Bierman EL. 2006. Atheroma and Other Forms of Atherosclerosis, in Isselbacher

KJ. Circulation. New York: McGraw Hill, pp 1106-116.

Corti, R.; Flammer, A.J.; Hollenberg, N.K. 2009. Cocoa and Cardiovascular

Health. Circulation. Contemporary Reviews in Cardiovascular Medicine

119: 1433–1441

Cho, H.J., Shashkin, P., Gleissner, C.A. et al. 2007. Physiological Genomics 29.

Nutritioin Research, 149-160.

Davignon J, Ganz P. 2013. Role of endothelial dysfunction in atherosclerosis.

Circulation 109(23 Suppl 1):III27-32

Davis, N.E. 2005. Atherosclerosis-an inflammatory process. J Insur Med. 37:

72-75

xiv

Departemen Kesehatan RI. 2003. Survei Kesehatan Nasional 2001: Laporan Studi

Mortalitas 2001: Pola penyakit penyebab kematian di Indonesia. Jakarta:

Badan Penelitian dan Pengembangan Kesehatan

Direktorat Jenderal Perkebunan. 2010. Pedoman teknis pembudidayaan buah

kakao di Indonesia. Jakarta: Departemen Pertanian.

Engler MB, Engler MM, Chen CY et al. 2004. Flavonoid-rich dark chocolate

improves endothelial function and increases plasma epicatechin

concentrations in healthy adults. J. Am. Coll. Nutr. 23:197-204.

Engler, M.B., Engler, M.M. 2006. The vasculoprotective effects of flavonoid-rich

cocoa and chocolate. Nutrition Research, 24(9) : 695-706

Faunci AS, Kasper DL, Braunwald E et al. 2008. Harrrison’s Principles of

Internal Medicine 17th Edition. New York: McGraw Hills

Fruchart JC, De Geteire C, Delfly B et al. 1994. Apolipoprotein A-I-containing

particles and reverse cholesterol transport: evidence for connection

between cholesterol efflux and atherosclerosis risk. Atherosclerosis 110

Suppl:S35-9.

Guyton Arthur C, Hall John E. 2006. Buku Ajar Fisiologi Kedokteran. Jakarta:

EGC, pp 1077-1088.

Grassi D, Necozione S, Lippi C et al. 2010. Cocoa reduces blood pressure and

insulin resistance and improves endothelium-dependent vasodilation in

hypertensives. Hypertension 46:398–405

Hanson, G.K. and Libby, P. 2009. The immune response in atherosclerosis: a

double-edged sword. Nature. 6: 508-519.

Heiss C, Kleinbongard P, Dejam A et al. 2009. Acute consumption of flavanol-

rich cocoa and the reversal of endothelial dysfunction in smokers. J Am

Coll Cardiol 46(7):1276–1283

Japardi I. 2002. Patofisiologi Stroke Infark Akibat Tromboemboli. Available from

URL: http://www.library.usu.ac.id diakses 28 September 2013.

xv

Lee MY & Griendling KK. 2008. Redox signaling, vascular function, and

hypertension. Antioxid Redox Signal 10, pp 1045–1059.

Libby, P. 2002. Inflammation in atherosclerosis. Nature. 420: 868-874.

Karmawati E, Mahmud Z, Syakir M et al. 2010. Budidaya dan Pasca Panen

Kakao. Pusat Penelitian dan Pengembangan Perkebunan Bogor, hal 10-21.

Keen CL, Holt RR, Oteiza PI et al. 2005. Cocoa antioxidants and cardiovascular

health. J Clin Nutr 81(1 Suppl):298S–303S

Manach C, Scalbert A, Morand C et al. 2009. Polyphenols: food sources and

bioavailability. Am J Clin Nutr;79:727-47.

Matsuda Y, Hirata K, Inoue N et al. 2003. High density lipoproteins reverses

inhibitory effect of oxidized low density lipoprotein on endothelium

dependent relaxation. Circ Res 72:1103-1109

Murphy KJ, Chronopoulos AK, Singh I, et al. 2003. Dietary flavanols and

procyanidin oligomers from cocoa (Theobroma cacao) inhibit platelet

function. Am J Clin Nutr 77(6):1466-1473.

Paoletti R, Poli A, Conti A et al. 2011. Chocolate and Health. Springer-Verlag

Italia; e-ISBN 978-88-470-2038-2: pp 1-153.

Peter, L. 2002. Inflammation in Atherosclerosis. Cardiovascular Division,

Department of Medicine, Brigham and Women’s Hospital, and Harvard

Medical School, Boston, Massachusetts 02115, USA.

Price Sylvia A, Wilson Lorraine M. 2005. Patofisiologi Konsep Klinis Proses-

Proses Penyakit. Ed 6. Jakarta: EGC

Rimbach G, Melchin M, Moehring J et al. 2009. Polyphenols from Cocoa and

vascular health-a critical review. In J Mol Sci 10: 4290–4309

Sargowo D. 2003. Pertemuan Ilmiah Nasional Reguler II Patobiologi : Peran

Lipid dan Radikal Bebas pada Patogenesa Aterosklerosis. Malang :

Perhimpunan Patobiologi Indonesia Cabang Malang dan FKUB

Schiffrin EL, Park JB, Intengan HD et al. 2004. Correction of arterial structure

and endothelial dysfunction in human essential hypertension. Circulation

101: 1653–1659

Silbernagl S, Florian L. 2006. Atlas Patofisiologi, hal 238. Jakarta: EGC.

xvi

Supranto, J. 2007. Teknik Sampling Survey & Eksperimen. Jakarta: PT Rineka.

Vallance P, Collier J, Moncada S. 2001. Nitric oxide synthesised from L-arginine

mediates endothelium dependent dilatation in human veins in vivo.

Cardiovasc Res 23(12):1053–1057.

Valenti VE, Abreu LC, Saldiva PH, Carvalho TD, Ferreira C. 2010. Effects of

sidestream cigarette smoke exposure on baroreflex components in

spontaneously hypertensive rats. Int J Environ. Health Res 20(6):431–7.

Vinson JA, Proch J, Bose P et al. 2006. Chocolate is a powerful ex vivo and in

vivo antioxidant, an antiatherosclerotic agent in an animal model, and a

significant contributor to antioxidants in the European and American Diets.

J Agric Food Chem 54:8071–8076

World Health Organization. 2001. WHO World Health Organization Report 2000,

Genewa: WHO.

Yasa, M. 2005. Vasoprotective Effects of Nitric Oxide in Atherosclerosis. FABAD

J. Pharm. Sci., 30, 41-5