Antihypercholesterolemic and Antioxidative Potential of an Extract of the Plant, Piper betle, and Its Active Constituent, Eugenol, in Triton WR-1339-Induced Hypercholesterolemia in Experimental Rats
Hypercholesterolemia is a dominant risk factor for atherosclerosis and cardiovascular diseases. In the present study, the putative antihypercholesterolemic and antioxidative properties of an ethanolic extract of Piper betle and of its active constituent, eugenol, were evaluated in experimental hypercholesterolemia induced by a single intraperitoneal injection of Triton WR-1339 (300？mg/kg？b.wt) in Wistar rats. Saline-treated hypercholesterolemic rats revealed significantly higher mean blood/serum levels of glucose, total cholesterol, triglycerides, low density and very low density lipoprotein cholesterol, and of serum hepatic marker enzymes; in addition, significantly lower mean serum levels of high density lipoprotein cholesterol and significantly lower mean activities of enzymatic antioxidants and nonenzymatic antioxidants were noted in hepatic tissue samples from saline-treated hypercholesterolemic rats, compared to controls. However, in hypercholesterolemic rats receiving the Piper betle extract (500？mg/kg？b.wt) or eugenol (5？mg/kg？b.wt) for seven days orally, all these parameters were significantly better than those in saline-treated hypercholesterolemic rats. The hypercholesterolemia-ameliorating effect was better defined in eugenol-treated than in Piper betle extract-treated rats, being as effective as that of the standard lipid-lowering drug, lovastatin (10？mg/kg？b.wt). These results suggest that eugenol, an active constituent of the Piper betle extract, possesses antihypercholesterolemic and other activities in experimental hypercholesterolemic Wistar rats. 1. Introduction Hypercholesterolemia is a major socioeconomic problem in common individuals as well as health professionals due to the strong correlation between cardiovascular diseases and lipid abnormalities [1]. The modern lifestyle, with a high fat diet and little physical activity, significantly contributes to hypercholesterolemia and cardiovascular diseases [2]. High levels of low-density lipoprotein (LDL) cholesterol accumulate in the extracellular subendothelial space of arteries; these are highly atherogenic and toxic to vascular cells, leading to atherosclerosis, hypertension, obesity, diabetes, and functional depression in organs such as the liver, heart, and kidneys [3]. Clinical trials have shown that lowering lipids reduces the morbidity and mortality associated with cardiovascular complications [4]. Intensive reduction of LDL-cholesterol levels have also been found to reverse atherosclerosis and decrease the progression of cardiovascular disease [5, 6]. Oxidative stress induced
References
[1]
M. A. Morsy and A. A. Fouad, “Mechanisms of gastroprotective effect of eugenol in indomethacin-induced ulcer in rats,” Phytotherapy Research, vol. 22, no. 10, pp. 1361–1366, 2008.
[2]
J. E. Freedman, “High-fat diets and cardiovascular disease: are nutritional supplements useful?” Journal of the American College of Cardiology, vol. 41, no. 10, pp. 1750–1752, 2003.
[3]
K. S. Jain, R. R. Kulkarni, and D. P. Jain, “Current drug targets for antihyperlipidemic therapy,” Mini Reviews in Medicinal Chemistry, vol. 10, no. 3, pp. 232–262, 2010.
[4]
？. L. Amundsen, L. Ose, M. S. Nenseter, and F. Y. Ntanios, “Plant sterol ester-enriched spread lowers plasma total and LDL cholesterol in children with familial hypercholesterolemia,” American Journal of Clinical Nutrition, vol. 76, no. 2, pp. 338–344, 2002.
[5]
T. Ichihashi, M. Izawa, K. Miyata, T. Mizui, K. Hirano, and Y. Takagishi, “Mechanism of hypocholesterolemic action of S-8921 in rats: S-8921 inhibits ileal bile acid absorption,” Journal of Pharmacology and Experimental Therapeutics, vol. 284, no. 1, pp. 43–50, 1998.
[6]
W. Shi, M. E. Haberland, M.-L. Jien, D. M. Shih, and A. J. Lusis, “Endothelial responses to oxidized lipoproteins determine genetic susceptibility to atherosclerosis in mice,” Circulation, vol. 102, no. 1, pp. 75–81, 2000.
[7]
D. L. Tribble, “Antioxidant consumption and risk of coronary heart disease: emphasis on vitamin C, vitamin E, and β-carotene: a statement for healthcare professionals from the American Heart Association,” Circulation, vol. 99, no. 4, pp. 591–595, 1999.
[8]
E. Hopps, D. Noto, G. Caimi, and M. R. Averna, “A novel component of the metabolic syndrome: the oxidative stress,” Nutrition, Metabolism and Cardiovascular Diseases, vol. 20, no. 1, pp. 72–77, 2010.
[9]
M. M. M. F. Duarte, J. B. T. Rocha, R. N. Moresco et al., “Association between ischemia-modified albumin, lipids and inflammation biomarkers in patients with hypercholesterolemia,” Clinical Biochemistry, vol. 42, no. 7-8, pp. 666–671, 2009.
[10]
I. Ahmad, Z. Mehmood, and F. Mohammad, “Screening of some Indian medicinal plants for their antimicrobial properties,” Journal of Ethnopharmacology, vol. 62, no. 2, pp. 183–193, 1998.
[11]
R. S. Koff, G. Gordan, and S. M. Sabesin, “D-Galactosamine hepatitis hepatocellular injury and fatty liver following a single dose,” Proceedings of the Society for Experimental Biology and Medicine, vol. 137, pp. 696–701, 1971.
[12]
D. Chakraborty and B. Shah, “Antimicrobial, anti-oxidative and anti-hemolytic activity of Piper betel leaf extracts,” International Journal of Pharmacy and Pharmaceutical Sciences, vol. 3, no. 3, pp. 192–199, 2011.
[13]
E. Nagababu and N. Lakshmaiah, “Inhibitory effect of eugenol on non-enzymatic lipid peroxidation in rat liver mitochondria,” Biochemical Pharmacology, vol. 43, no. 11, pp. 2393–2400, 1992.
[14]
H. Kabuto, M. Tada, and M. Kohno, “Eugenol [2-methoxy-4-(2-propenyl)phenol] prevents 6-hydroxydopamine-induced dopamine depression and lipid peroxidation inductivity in mouse striatum,” Biological and Pharmaceutical Bulletin, vol. 30, no. 3, pp. 423–427, 2007.
[15]
World Health Organization, “FAO/WHO expert committee on food additives evaluation of certain food additives and contaminants,” Tech. Rep. 683, WHO, Geneva, Switzerland, 1982.
[16]
A. M. Rimando, B. H. Han, J. H. Park, and M. C. Cantoria, “Studies on the constituents of Philippine Piper betle leaves,” Archives of Pharmacal Research, vol. 9, no. 2, pp. 93–97, 1986.
[17]
A. Manigauha, S. Patel, H. Ali, A. Chandy, and M. Uma Maheshwari, “Study the effect of phytochemical constituents of Piper betle leaves extracts on liver disorders by in vivo model,” Journal of Pharmacy Research, vol. 2, pp. 353–356, 2009.
[18]
P. E. Schurr, J. R. Schultz, and T. M. Parkinson, “Triton-induced hyperlipidemia in rats as an animal model for screening hypolipidemic drugs,” Lipids, vol. 7, no. 1, pp. 68–74, 1972.
[19]
M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976.
[20]
T. Sasaki, S. Matzy, and A. Sonal, “Effect of acetic acid concentration on the colour reaction in the O-toluidine boric acid method for blood glucose estimation,” Rinsho Kagaku, vol. 1, pp. 346–353, 1972.
[21]
W. T. Friedewald, R. I. Levy, and D. S. Fredrickson, “Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge,” Clinical Chemistry, vol. 18, no. 6, pp. 499–502, 1972.
[22]
J. King, “The transferases-alanine and aspartate transaminases,” in Practical Clinical Enzymology, D. Van, Ed., pp. 121–138, D. Van Nostrand, London, UK, 1965.
[23]
J. King, “The hydrolases-acid and alkaline phosphatases,” in Practical Clinical Enzymology, D. Van, Ed., pp. 191–208, D. Van Nostrand, London, UK, 1965.
[24]
J. King, “The dehydrogenases or oxidoreductases lactate dehydrogenase,” in Practical Clinical Enzymology, D. Van, Ed., D. Van Nostrand, London, UK, 1965.
[25]
A. K. Sinha, “Colorimetric assay of catalase,” Analytical Biochemistry, vol. 47, no. 2, pp. 389–394, 1972.
[26]
S. Marklund and G. Marklund, “Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase,” European Journal of Biochemistry, vol. 47, no. 3, pp. 469–474, 1974.
[27]
J. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman, and W. G. Hoekstra, “Selenium: biochemical role as a component of glatathione peroxidase,” Science, vol. 179, no. 4073, pp. 588–590, 1973.
[28]
W. H. Habig and W. B. Jakoby, “[51] Assays for differentiation of glutathione S-Transferases,” Methods in Enzymology, vol. 77, pp. 398–405, 1981.
[29]
M. S. Moron, J. W. Depierre, and B. Mannervik, “Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver,” Biochimica et Biophysica Acta, vol. 582, no. 1, pp. 67–78, 1979.
[30]
S. T. Omaye, J. David Turnbull, and H. E. Sauberlich, “[1] Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids,” Methods in Enzymology, vol. 62, pp. 3–11, 1979.
[31]
I. D. Desai, “Vitamin E analysis methods for animal tissues,” Methods in Enzymology, vol. 105, pp. 138–147, 1984.
[32]
H. Ohkawa, N. Ohishi, and K. Yagi, “Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction,” Analytical Biochemistry, vol. 95, no. 2, pp. 351–358, 1979.
[33]
R. A. B. Drury and E. A. Wallington, Wallington, Carleton’s Histological Technique, Oxford University Press, New York, NY, USA, 1980.
[34]
S. O. Byers, M. Fiedman, and T. Sugiyama, “Triton hypercholesteremia: cause or consequence of augmented cholesterol synthesis,” The American Journal of Physiology, vol. 204, pp. 1100–1102, 1963.
[35]
H. Harnafi, H. Serghini Caid, N. el Houda Bouanani, M. Aziz, and S. Amrani, “Hypolipemic activity of polyphenol-rich extracts from Ocimum basilicum in Triton WR-1339-induced hyperlipidemic mice,” Food Chemistry, vol. 108, no. 1, pp. 205–212, 2008.
[36]
A. Ansarullah, R. Jadeja, M. Thounaojam, V. Patel, R. Devkar, and A. Ramachandran, “Antihyperlipidemic potential of a polyherbal preparation on Triton WR 1339 (Tyloxapol) induced hyperlipidemia: a comparison with lovastatin,” International Journal of Green Pharmacy, vol. 3, no. 2, pp. 119–124, 2009.
[37]
P. Vijayaraj, K. Muthukumar, J. Sabarirajan, and V. Nachiappan, “Antihyperlipidemic activity of Cassia auriculata flowers in triton WR 1339 induced hyperlipidemic rats,” Experimental and Toxicologic Pathology, no. 1-2, pp. 135–141, 2013.
[38]
S.-J. Lee, J.-H. Ko, K. Lim, and K.-T. Lim, “150 kDa glycoprotein isolated from Solanum nigrum Linne enhances activities of detoxicant enzymes and lowers plasmic cholesterol in mouse,” Pharmacological Research, vol. 51, no. 5, pp. 399–408, 2005.
[39]
G. Shattat, R. Al-Qirim, Y. Al-Hiari et al., “Synthesis and anti-hyperlipidemic evaluation of N-(benzoylphenyl)-5-fluoro- 1H-indole-2-carboxamide derivatives in Triton WR-1339-induced hyperlipidemic rats,” Molecules, vol. 15, no. 9, pp. 5840–5849, 2010.
[40]
G. Shattat, T. Al-Qirim, K. Sweidan, M. Shahwan, W. El-Huneidi, and Y. Al-Hiari, “The hypolipidemic activity of novel benzofuran-2-carboxamide derivatives in Triton WR-1339-induced hyperlipidemic rats: a comparison with bezafibrate,” Journal of Enzyme Inhibition and Medicinal Chemistry, vol. 25, no. 6, pp. 751–755, 2010.
[41]
J. J. ？gren, J.-P. Kurvinen, and A. Kuksis, “Isolation of very low density lipoprotein phospholipids enriched in ethanolamine phospholipids from rats injected with Triton WR 1339,” Biochimica et Biophysica Acta, vol. 1734, no. 1, pp. 34–43, 2005.
[42]
P. R. Holt and A. A. Dominguez, “Triton-induced hyperlipidemia: a model for studies of intestinal lipoprotein production,” The American Journal of Physiology, vol. 238, no. 5, pp. G453–G457, 1980.
[43]
A. K. Khanna, F. Rizvi, and R. Chander, “Lipid lowering activity of Phyllanthus niruri in hyperlipemic rats,” Journal of Ethnopharmacology, vol. 82, no. 1, pp. 19–22, 2002.
[44]
G. Davey Smith and J. Pekkanen, “Should there be a moratorium on the use of cholesterol lowering drugs?” British Medical Journal, vol. 304, no. 6824, pp. 431–434, 1992.
[45]
M. J. Malloy and J. P. Kane, “Medical management of hyperlipidemic states,” Advances in Internal Medicine, vol. 39, pp. 603–631, 1994.
[46]
J. E. Hokanson and M. A. Austin, “Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a meta-analysis of population-based prospective studies,” Journal of Cardiovascular Risk, vol. 3, no. 2, pp. 213–219, 1996.
[47]
M. A. Austin, J. E. Hokanson, and J. D. Brunzell, “Characterization of low-density lipoprotein subclasses: methodologic approaches and clinical relevance,” Current Opinion in Lipidology, vol. 5, no. 6, pp. 395–403, 1994.
[48]
T. Murakami, S. Michelagnoli, R. Longhi et al., “Triglycerides are major determinants of cholesterol esterification/transfer and HDL remodeling in human plasma,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 15, no. 11, pp. 1819–1828, 1995.
[49]
M. J. Thomas, “Physiological aspects of low density lipoprotein oxidation,” Current Opinion in Lipidology, vol. 11, pp. 297–301, 2000.
[50]
I. Vallianou, N. Peroulis, P. Pantazis, and M. Hadzopoulou-Cladaras, “Camphene, a plant-derived monoterpene, reduces plasma cholesterol and triglycerides in hyperlipidemic rats independently of HMG-CoA reductase activity,” PLoS ONE, vol. 6, no. 11, Article ID e20516, 2011.
[51]
C. Kü？ükgergin, A. F. AydIn, G. ？zdemirler-Erata, G. Mehmet？ik, N. Ko？ak-Toker, and M. Uysal, “Effect of artichoke leaf extract on hepatic and cardiac oxidative stress in rats fed on high cholesterol diet,” Biological Trace Element Research, vol. 135, no. 1–3, pp. 264–274, 2010.
[52]
D. W. Molander, F. Wroblewski, and J. S. ladue, “Serum glutamic oxalacetic transaminase as an index of hepatocellular integrity,” Journal of Laboratory and Clinical Medicine, vol. 6, pp. 831–839, 1955.
[53]
R. Anandhi, T. Annadurai, T. S. Anitha et al., “Antihypercholesterolemic and antioxidative effects of an extract of the oyster mushroom, Pleurotus ostreatus, and its major constituent, chrysin, in Triton WR-1339-induced hypercholesterolemic rats,” Journal of Physiology and Biochemistry, vol. 69, no. 2, pp. 313–323, 2013.
[54]
K. Prasad, “Reduction of serum cholesterol and hypercholesterolemic atherosclerosis in rabbits by secoisolariciresinol diglucoside isolated from flaxseed,” Circulation, vol. 99, no. 10, pp. 1355–1362, 1999.
[55]
B. Halliwell, “Lipid peroxidation, antioxidants and cardiovascular disease: how should we move forward?” Cardiovascular Research, vol. 47, no. 3, pp. 410–418, 2000.
[56]
C. G. Sheela and K. T. Augusti, “Antiperoxide effects of S-allyl cysteine sulphoxide isolated from Allium sativum Linn and gugulipid in cholesterol diet fed rats,” Indian Journal of Experimental Biology, vol. 33, no. 5, pp. 337–341, 1995.
[57]
C. D. Klaassen, W. M. Bracken, R. E. Dudley, P. L. Goering, G. A. Hazelton, and J. J. Hjelle, “Role of sulfhydryls in the hepatotoxicity of organic and metallic compounds,” Toxicological Sciences, vol. 5, no. 5, pp. 806–815, 1985.
[58]
M. G. Traber and H. Sies, “Vitamin E in humans: demand and delivery,” Annual Review of Nutrition, vol. 16, pp. 321–347, 1996.
[59]
B. A. Freeman and J. D. Crapo, “Biology of disease. Free radicals and tissue injury,” Laboratory Investigation, vol. 47, no. 5, pp. 412–426, 1982.
[60]
K. Prasad and J. Kalra, “Oxygen free radicals and hypercholesterolemic atherosclerosis: effect of vitamin E,” American Heart Journal, vol. 125, no. 4, pp. 958–973, 1993.
[61]
E. Niki, H. Shimasaki, and M. Mino, Antioxidant-Free Radical and Biological Defence, Gakkai-Shuppan Center, Tokyo, Japan, 1994.
[62]
P. R. Deepa and P. Varalakshmi, “Protective effects of certoparin sodium, a low molecular weight heparin derivative, in experimental atherosclerosis,” Clinica Chimica Acta, vol. 339, no. 1-2, pp. 105–115, 2004.
[63]
V. Sudhahar, S. A. Kumar, and P. Varalakshmi, “Role of lupeol and lupeol linoleate on lipemic-oxidative stress in experimental hypercholesterolemia,” Life Sciences, vol. 78, no. 12, pp. 1329–1335, 2006.
