Sains Malaysiana 49(1)(2020): 93-101

http://dx.doi.org/10.17576/jsm-2020-4901-11

Protective Effect of Cocoa Extract on Ethanol Induced Liver Injury in Sprague-Dawley Rats

(Kesan Pelindung Ekstrak Koko untuk Kecederaan Hati Teraruh Etanol pada Tikus Sprague-Dawley)

ROSMAWATI MAT SHAIR^1,2, MOHAMAD YUSOF MASKAT²*, MOHAMAD KHAN AYOB² & ROSMIN KASRAN¹

¹Division of Biotechnology, Cocoa Innovation and Technology Centre, Malaysia Cocoa Board, Nilai Industrial Park, 71800 Nilai, Negeri Sembilan Darul Khusus, Malaysia

²Center of Biotechnology & Functional Food, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

Received: 19 June 2019/Accepted: 15 October 2019

ABSTRACT

Cocoa is a rich source of dietary polyphenol, highly potential antioxidant against free radicals. This study was designed to identify the effect of cocoa polyphenol extract in protecting from ethanol-induced liver injury in rats. Fifty male Sprague-dawley rats were divided into five groups fed with or without ethanol (4 g/kg/d), cocoa extract (300 mg/kg/d) and silymarin (200 mg/kg/d) continuously for 3 weeks using an enteral feeding protocol. All treatments were given orally every day for three weeks and continuously supply food and water ad libitum. Results showed that cocoa extract (CE) from unfermented cocoa beans had a total polyphenol content of 335.70±27.51 mg GAE/g and 38.10±4.52 mg CaE/g. Meanwhile, analysis normal phase-high performance liquid chromatography shows CE contains 59.47±9.44 mg/g and 14.69±1.63 mg/g of epicathechin and catechin, respectively, which is three fold higher compared to commercial cocoa powder. It also contains 59.69±2.15 mg/g theobromine which also three fold higher compared to caffeine 19.87±1.37 mg/g. In vitro study showed cocoa extract contains high antioxidant activities by 91.9±1.00 % against superoxide scavenging system (O₂^-) and 97.7±0.15% against  a-a-diphenyl-β-picrylhydrazyl radical (DPPH) systems. In vivo study showed increasing level in both liver function enzymes, aspartase aminotransferase (AST) and alanine aminotransferase (ALT) in ethanol intoxication by 116.80±5.23 mmol/L and 56.37±2.71 mmol/L, respectively. Ethanol intoxication was blocked by cocoa extract nearly 89.95±1.18 mmol/L and 46.75±0.74 mmol/L, respectively, and it was comparable with SDT group for both enzymes AST and ALT by 112.19±6.02 mmol/L and 42.49±0.62 mmol/L, respectively. Furthermore, ethanol groups showed significantly lower (p<0.05) of glutathione level by 0.29 ±0.03 µmol/g, however cocoa extract with antioxidant defense system either direct or indirectly protect liver injury by increasing glutathione level at 0.53±0.02 µmol/g. As a result, cocoa extract shows its potential as antioxidant agents to protect ethanol-induced liver injury.  

Keywords: Antioxidant activity; chronic ethanol; cocoa extract; glutathione; liver injury

ABSTRAK

Koko kaya dengan sumber polifenol diet bertindak sebagai antioksida dan berpotensi untuk menyingkirkan radikal bebas. Kajian ini dilakukan bagi mengenal pasti kesan ekstrak polifenol daripada koko untuk mencegah kerosakan hati tikus yang disebabkan oleh pengambilan etanol. Sebanyak lima puluh ekor tikus Sprague-dawley jantan, dibahagikan kepada lima kumpulan iaitu kumpulan kawalan, etanol (4 g/kg/d), ekstrak koko (300 mg/kg/d) dan silimarin (200 mg/kg/d) yang mengandungi sepuluh ekor bagi setiap kumpulan. Setiap rawatan diberikan secara oral selama 3 minggu dan pengambilan makanan dan minuman adalah secaraad libitum. Analisis Folin-ciocalteau ekstrak koko (CE) telah menunjukkan bahawa jumlah kandungan polifenol ialah sebanyak 335.70±27.51 mg GAE/g dan 38.10±4.52 mg CaE/g. Manakala keputusan fasa normal kromatografi cecair berprestasi tinggi (NP-HPLC) pula menunjukkan ekstrak koko mengandungi kandungan epikatekin sebanyak59.47±9.44 mg/g dan katekin sebanyak 14.69±1.63 mg/g iaitu tiga kali ganda lebih tinggi berbanding serbuk koko komersial. Ia juga mengandungi theobromina sebanyak 59.69±2.15 mg/g, iaitu tiga kali ganda kandungan lebih tinggi berbanding kafein iaitu 19.87±1.37 mg/g. Kajianin vitro menunjukkan aktiviti antioksida ektrak koko adalah sangat tinggi iaitu sebanyak 91.9±1.00% terhadap radikal superoksida (O₂^-) dan 97.7±0.15% terhadap radikala-a-difenil-β-pikrilhidrazil (DPPH). Manakala kajianin vivo menunjukkan berlaku peningkatan terhadap kedua-dua enzim fungsi hati iaitu enzim aspartase aminotransferase (AST) dan alanine aminotransferase (ALT) di dalam ketoksikan etanol masing-masing sebanyak 116.80±5.23 mmol/L dan 56.37±2.71 mmol/L. Ketoksikan etanol dapat dicegah dengan kahadiran ekstrak koko dengan kandungan enzim masing-masing adalah sebanyak 89.95±1.18 mmol/L dan 46.75±0.74 mmol/L, selari dengan kumpulan silimarin bagi kedua-dua enzim AST dan ALT iaitu masing-masing 112.19±6.02 mmol/L dan 42.49±0.62 mmol/L. Selanjutnya, kumpulan etanol menunjukkan penurunan yang signifikan (P<0.05) kandungan glutation sejenis antioksida pertahanan badan semula jadi sebanyak 0.29 ±0.03 µmol/g, walau bagaimanapun, kehadiran ekstrak koko yang bertindak samada secara langsung atau tidak langsung melindungi kerosakan hati telah meningkatkan kandungan glutation sebanyak 0.53±0.02 µmol/g. Kesimpulannya, ekstrak koko telah menunjukkan bahawa ia sangat berpotensi sebagai agen antioksida dalam melindungi kerosakan hati yang disebabkan oleh pengambilan etanol.

Kata kunci: Aktiviti antioksida; ekstrak koko; etanol kronik; glutation; kerosakan hati

REFERENCES

Adam, W.B. 1928. Determination of the color-producing constituents of the cacao bean. The Analyst 53: 369-372.

Ashihara, H., Sano, H. & Crozier, A. 2008. Caffeine and related purine alkaloids: Biosynthesis, catabolism, function and genetic engineering. Phytochemistry 64(4): 841-856.

Blois, M.S. 1958. Antioxidant determinations by the use of a stable free radical. Nature 181: 1199-1200.

Caballeria, J. 2003. Current concepts in ethanol metabolism. Ann. Hepatol. 2: 60-68.

Cienfuegos-Jovellanos, E., Del Mar, Q., Muguerza, B., Moulay, L., Miguel, M. & Aleixandre, A. 2009. Antihypertensive effect of a polyphenol-rich cocoa powder industrially processed to preserve the original flavonoids of the cocoa beans. J. Agric. Food Chem. 57: 6156-6162.

Coleman, R.A. 1980. Purine antagonists in the identification of adenosine-receptors in guinea-pig trachea and the role of purines in none-adrenergic inhibitory neurotransmission. Br. J. Pharmacol. 69: 359-366.

Costa, J., Lunet, N., Santos, C., Santos, J. & Vaz-Carneiro, A. 2010. Caffeine exposure and the risk of Parkinson’s disease: A systematic review and meta-analysis of observational studies. J. Alzheimer’s Dis. 20: S221–S238.

Davies, K.J. 1987. Protein damage and degradation & oxygen radicals. I. General aspects. Journal of Biological Chemistry 262: 9895-9901.

Davies, K.J. & Delsignore, M.E. 1987. Protein damage and degradation by oxygen radicals. III. Modification of secondary and tertiary structure. Journal of Biological Chemistry 262: 9908-9913.

Ellman, G.L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82: 70-77.

Eskelinen, M.H., Ngandu, T., Tuomilehto, J., Soininen, H. & Kivipelto, M. 2009. Midlife coffee and tea drinking and the risk of late-life dementia: A population-based CAIDE study. Journal of Alzheimer's Disease 16: 85-91.

Fraga, C.G. 2007. Plant polyphenols: How to translate their in vitro antioxidant actions to in vivo conditions. IUBMB Life 59: 308-315.

Franco, R., Oñatibia-Astibia, A. & Martínez-Pinilla, E. 2013. Health benefits of methylxanthine in cacoa and chocolate. Nutrients 5: 4159-4173.

Haller, S., Rodriguez, C., Moser, D., Toma, S., Hofmeister, J., Sinanaj, I., Van der ville, D., Giannakopoulos, P. & Lovblad, K.O. 2013. Acute caffeine administration impact on working memory-related brain activation and functional connectivity in the elderly: A BOLD and perfusion MRI study. Neuroscience 250: 364-371.

Halliwell, B. & Gutteridge, J.M.C. 1999. Chapter 4: Antioxidants from the diet. In Free Radicals in Biology and Medicine. 3rd ed. Oxford: Clarendon Press.

Hammerstone, J.F., Lazarus, S.A., Mitchel, A.E., Rucker, R. & Schmitz, H.H. 1999. Identification of procyanidins in cocoa (Theobroma Cacao) and chocolate using high-performance liquid chromatography/mass spectrometry. Journal of Agricultural Food Chemistry 47: 490-496. 

Han, M.E., Kim, H.J., Lee, Y.S., Kim, D.H., Choi, J.T., Pan, C.S., Yoon, S., Baek, S.Y., Kim, B.S. & Kim, J.B. 2009. Regulation of cerebral fluid production by caffeine consumption. BMC Neuroscience 10: 110.

Hingson, R. & Howland, J. 2002. Comprehensive community interventions to promote health: Implication for college-age drinking problems. J. Stud. Alcohol. 14: 226-240.

Hingson, R., Heeren, T., Winter, M. & Wechsler, H. 2005. Magnitude of alcohol-related mortality and morbidity among U.S college students ages 18-24: Changes from 1998 to 2001. Annu. Rev. Public Health 26: 259-279.

Hollman, P.C.H., Cassidy, A., Comte, B., Heinonen, M., Richelle, M., Richling, E., Serafini, M., Scalbert, A., Sies, H. & Vidry, S. 2011. The biological relevance of direct antioxidant effects of polyphenols for cardiovascular health in humans is not established. The Journal of Nutrition 141: 989S-1009S.

Ighodaro, O.M. & Akinloye, O.A. 2018. First line defence antioxidant-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid. Alexandria Journal of Medicine 54(4): 287-293.

Kerimi, A. & Williamson, G. 2015. The cardiovascular benefits of dark chocolate. Vascular Pharmacology 71: 11-15.

Khan, N., Monagas, M., Andres-Lacueva, C., Casas, R., Urpí-Sardà, M., Lamuela-Raventós, R.M. & Estruch, R. 2012. Regular consumption of cocoa powder with milk increases HDL cholesterol and reduced oxidized LDL levels in subjects at high-risk of cardiovascular disease. Nutr. Metab. Cardiovasc. Dis. 22: 1046-1053.

Kono, Y. & Fridovich, I. 1982. Superoxide radical inhibits catalase. Journal of Biological Chemistry 257: 5751-5754.

Koppelstaetter, F., Poeppel, T.D., Siedentopf, C.M., Ischebeck, A., Verius, M., Haala, I., Mottaghy, F.M., Rhomberg, P., Golaszewski, S. & Gotwald, T. 2008. Does caffeine modulate verbal memory working processes? An fMRI study. NeuroImage 39: 492-499.

Lambert, J.D. & Elias, R. 2010. The antioxidant and pro-oxidant activities of green tea polyphenols: A role in cancer prevention. Archieves of Biochemistry and Biophysics 501: 65-72.

Lieber, C.C. 1997. Role of oxidative stress and antioxidant therapy in alcoholic and nonalcoholic liver disease. Advanced Pharmacology 38: 601-628.

Maia, L. & de Mendonca, A. 2002. Does caffeine intake protect from Alzheimer’s disease? Eur. J. Neurol. 9: 377-382.

Martin, M.A., Goya, L. & Ramos, S. 2016. Antidiabetic actions of cocoa flavonols. Molecular Nutrition and Food Research doi:10.1002/mnfr.201500961.

Martin, M.A., Goya, L. & Ramos, S. 2013. Potential for preventive effect of cocoa and cocoa polyphenols in cancer. Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association 56: 336-351.

Mauch, T.J., Donohue Jr., T.M., Zetterman, R.K., Sorrel, M.F. & Tuma, D.J. 1986. Covalent binding of acetaldehyde selectively inhibits the catalytic activity of lysine-dependent enzymes. Hepatology 6: 263-269.

Nakagawa, H., Hasumi, K., Woo, J., Nagai, K. & Wachi, M. 2004. Generation of hydrogen peroxide primarily contributes to the induction of Fe(II)-dependent apoptosis in Jurkat cells by (-)-epigallocatechin gallate. Carcinogenesis 25: 1567-1574.

Nehlig, A. 2010. Is caffeine a cognitive enhancer? J. Alzheimer’s Dis. 2: S85-S94.

Neufingerl, N., Zebregs, Y.E., Schuring, E.A. & Trautwein, E.A. 2013. Effect of cocoa and theobromine consumption on serum HDL-cholesterol concentration: A randomized controlled trial. Am. J. Clin. Nutr. 97: 1201-1209.

Nordman, R. 1994. Alcohol and antioxidant system. Alcoholism and Alcohol 29: 513-522.

Orru, M., Bakešová, J., Brugarolas, M., Quiroz, C., Beaumont, V., Goldberg, S.R., Lluís, C., Cortés, A., Franco, R. & Casadó, V. 2011. Striatal pre- and postsynaptic profile of adenosine A2A receptor antagonists. PLoS ONE 6: e16088.

Othman, A., Amin, I., Nawalyah, A.G. & Ilham, A. 2007. Antioxidant capacity and phenolic content of cocoa beans. Food Chemistry 100(4): 1523-1530.

Pelligrino, D.A., Xu, H.L. & Vetri, F. 2010. Caffeine and the control of cerebral hemodynamics. J. Alzheimer's Dis. 20: S51-S62.

Pereira-Caro, G., Borges, G., Nagai, C., Jackson, M.C., Yakota, T., Crozier, A. & Ashihara, H. 2013. Profiles of phenolic compounds and purine alkaloids during the development of seeds of Theobroma cacao cv. Trinitario. Journal of Agricultural and Food Chemistry 61: 427-434.

Puntarulo, S., Stoyanovsky, D.A. & Cederbaum, A.I. 1999. Interaction of 1-hydroxyethyl radical with antioxidant enzymes. Archives of Biochemistry and Biophysics 15: 355-359.

Ramos, S. 2008. Cancer chemoprevention and chemotherapy: Dietary polyphenols and signaling pathways. Molecular Nutrition and Food Research 52: 507-526.

Rice-Evans C.A., Miller, N.J. & Paganga, G. 1997. Antioxidant properties of phenolic compounds. Trends Plant Sci. 2(4): 152-159.

Sallie, R., Tredger, J.M. & William, R. 1991. Drug and the liver. Biopharm. Drug Dispos. 12: 251-259.   

Samman, S., Sandström, B., Toft, M.B., Bukhave, K., Jensen, M. & Sorensen, S.S. 2001. Green tea or rosemary extract added to foods reduces nonheme-iron absorption. Am. J. Clin. Nutr. 73: 607-612.

Shahidi, F. & Ambigaipalan, P. 2015. Phenolics and polypenolics in foods, beverages and spices: Antioxidant activity and health effects-A review. Journal of Functional Foods 18: 820-897. 

Strubelt, O., Younes, M., Urch, T., Breining, H. & Pentz, R. 1987. Hepatotoxicity of acetaldehyde in rats. Toxicology Letter 39: 77-84.

Sussman, S., Dent, C.W., Skara, S., de Callice, P. & Tsukamoto, H. 2002. Alcoholic liver disease (ALD): A new domain for prevention effort. Subst. Use Misuse 37: 1887-1904.

Szabo, G.  2003. Pathogenic interactions between alcohol and hepatitis. C. Curr. Gastroenterol. Rep. 5: 86-92.

Usmani, O.S., Belvisim, M.G., Patel, H.J., Crispino, N., Birrell, M.A., Korbonits, D. & Barnes, P.J. 2005. Theobromine inhibits sensory nerve activation and cough. FAESB J. 19: 231-233.

Valko, M., Izakovic, M., Mazur, M., Rhodes, C.J. & Telser, J. 2004. Role of oxidation radicals in DNA damage and cancer incidence. Mol. Cell Biochem. 266: 37-56.

Vimala, S., Mohd Ilham, A., Rashih, A.A. & Rohana, S. 2003. Natural Antioxidants: Piper sarmentosum(Kadok) and Morinda elliptica(Mengkudu). Malaysia Journal of Nutrition 9(1): 41-51.

Wostyn, P., van Dam, D., Audenaert, K. & de Deyn, P.P. 2011. Increased cerebral fluid production as a possible mechanism underlying Caffein's protective effect against Alzheimer's disease. Int. J. Alzheimer's Dis. 2011: 617420. doi:10.4061/2011/617420.

*Corresponding author; email: yusufm@ukm.edu.my