title: Olive leaf extracts protect cardiomyocytes against 4-hydroxynonenal-induced toxicity in vitro: comparison with oleuropein, hydroxytyrosol, and quercetin.
abstract: Olive (Olea europaea) leaf, an important traditional herbal medicine, displays cardioprotection that may be related to the cellular redox modulating effects of its polyphenolic constituents. This study was undertaken to investigate the protective effect of the ethanolic and methanolic extracts of olive leaves compared to the effects of oleuropein, hydroxytyrosol, and quercetin as a positive standard in a carbonyl compound (4-hydroxynonenal)-induced model of oxidative damage to rat cardiomyocytes (H9c2). Cell viability was detected by the MTT assay; reactive oxygen species production was assessed by the 2',7'-dichlorodihydrofluorescein diacetate method, and the mitochondrial membrane potential was determined using a JC-1 dye kit. Phospho-Hsp27 (Ser82), phospho-MAPKAPK-2 (Thr334), phospho-c-Jun (Ser73), cleaved-caspase-3 (cl-CASP3) (Asp175), and phospho-SAPK/JNK (Thr183/Tyr185) were measured by Western blotting. The ethanolic and methanolic extracts of olive leaves inhibited 4-hydroxynonenal-induced apoptosis, characterized by increased reactive oxygen species production, impaired viability (LD50: 25 µM), mitochondrial dysfunction, and activation of pro-apoptotic cl-CASP3. The ethanolic and methanolic extracts of olive leaves also inhibited 4-hydroxynonenal-induced phosphorylation of stress-activated transcription factors, and the effects of extracts on p-SAPK/JNK, p-Hsp27, and p-MAPKAPK-2 were found to be concentration-dependent and comparable with oleuropein, hydroxytyrosol, and quercetin. While the methanolic extract downregulated 4-hydroxynonenal-induced p-MAPKAPK-2 and p-c-Jun more than the ethanolic extract, it exerted a less inhibitory effect than the ethanolic extract on 4-hydroxynonenal-induced p-SAPK/JNK and p-Hsp27. cl-CASP3 and p-Hsp27 were attenuated, especially by quercetin. Experiments showed a predominant reactive oxygen species inhibitory and mitochondrial protecting ability at a concentration of 1-10 µg/mL of each extract, oleuropein, hydroxytyrosol, and quercetin. The ethanolic extract of olive leaves, which contains larger amounts of oleuropein, hydroxytyrosol, verbascoside, luteolin, and quercetin (by HPLC) than the methanolic one, has more protecting ability on cardiomyocyte viability than the methanolic extract or each phenolic compound against 4-hydroxynonenal-induced carbonyl stress and toxicity.
authors.affiliation: Cellular Stress Response & Signal Transduction Research Laboratory, Department of Medical Pharmacology, Faculty of Medicine, Gazi University, and Farmasens Biotech Co., Gazi TechnoPark, Ankara, Turkey.
authors.forename: Elif Burcu
authors.initials: EB
authors.lastname: Bali
authors.affiliation: Department of Medical Biology & Genetics, Faculty of Medicine, Gazi University, Ankara, Turkey.
authors.forename: Volkan
authors.initials: V
authors.lastname: Ergin
authors.affiliation: Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovak Republic.
authors.forename: Lucia
authors.initials: L
authors.lastname: Rackova
authors.affiliation: Department of Chemical & Biochemical Engineering, Izmir Institute of Technology, and DUAG Co., İYTE, İzmir, Turkey.
authors.forename: Oğuz
authors.initials: O
authors.lastname: Bayraktar
authors.affiliation: Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara, Turkey.
authors.forename: Nurgün
authors.initials: N
authors.lastname: Küçükboyaci
authors.affiliation: Cellular Stress Response & Signal Transduction Research Laboratory, Department of Medical Pharmacology, Faculty of Medicine, Gazi University, and Farmasens Biotech Co., Gazi TechnoPark, Ankara, Turkey.
authors.forename: Çimen
authors.initials: Ç
authors.lastname: Karasu
mesh_terms: D000447:Aldehydes; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D066298:In Vitro Techniques; D057888:Iridoid Glucosides; D039823:Iridoids / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D031658:Olea / Q000737:chemistry*; D018384:Oxidative Stress / Q000187:drug effects*; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives / Q000494:pharmacology; D010936:Plant Extracts / Q000494:pharmacology*; D018515:Plant Leaves / Q000737:chemistry; D059808:Polyphenols / Q000494:pharmacology*; D020011:Protective Agents / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011794:Quercetin / Q000494:pharmacology; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D000447:Aldehydes; D000975:Antioxidants; D057888:Iridoid Glucosides; D039823:Iridoids; D010936:Plant Extracts; D059808:Polyphenols; D020011:Protective Agents; D014157:Transcription Factors; C005975:3,4-dihydroxyphenylethanol; C002769:oleuropein; D011794:Quercetin; D017346:Protein Serine-Threonine Kinases; D053148:Caspase 3; C027576:4-hydroxy-2-nonenal; D010626:Phenylethyl Alcohol