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
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.
i
dr. Kusuma Andriana, Sp.OG
Anggota
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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.
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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
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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
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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
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DAFTAR ISI
LEMBAR PENGESAHAN……………………………………………….
i
LEMBAR PENGUJIAN…………………………………………………..
ii
KATA PENGANTAR……………………………………………………..
iii
ABSTRAK………………………………………………………………....
v
ABSTRACT………………………………………………………………....
vi
DAFTAR ISI……………………………………………………………….
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DAFTAR SINGKATAN..............................................................................
x
DAFTAR GAMBAR....................................................................................
xi
DAFTAR TABEL ........................................................................................
BAB 1 PENDAHULUAN...........................................................................
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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
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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
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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
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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
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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`
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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
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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 apoAII 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
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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 flavanolrich 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.
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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 ProsesProses 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.
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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
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