AIDIC



CHEMICAL ENGINEERING TRANSACTIONS VOL. 76, 2019A publication ofThe Italian Associationof Chemical EngineeringOnline at aidic.it/cetGuest Editors: Sauro Pierucci, Ji?í Jaromír Kleme?, Laura PiazzaCopyright ? 2019, AIDIC Servizi S.r.l.ISBN 978-88-95608-73-0; ISSN 2283-9216Extraction and Characterization of the Phenolic Compounds from Leaves of Olea europaea L. via PLEJonas M. Jaskia, Tatiana C. Pimentelb, Carlos E. Bar?ob, Mayara S. Pontec, Lilian R. B. Mariuttic, Neura Bragagnoloc, Andressa C. Feihrmannd, Lucio Cardozo-Filhoa,e*aDepartment of Agronomy – State University of Maringa (UEM), Maringá, PR, BrazilbFederal Institute of Paraná (IFPR) - Paranavaí, Paraná, BrazilcDepartment of Food and Nutrition – School of Food Engineering – University of Campinas, Campinas, S?o Paulo, BrazildDepartment of Food Engineering - State University of Maringa (UEM), Maringá, PR, BrazileResearch Center – Centro Universitário Funda??o de Ensino Octávio Bastos (UNIFEOB) – S?o Jo?o da Boa Vista, Brazil lucio.cardozo@Oleuropein is the predominant phenolic compound in olive leaves. It presents high antioxidant and anti-inflammatory activities and has antimicrobial action. The objective of this study was to extract the phenolic compounds from dried olive leaves using pressurized ethanol in a continuous flow and characterize them. The flow rate (0.5 to 1.0 mL min-1), temperature (30 to 60 °C) and pressure (10 to 20 MPa) were the operational parameters. The results showed that the temperature was a significant factor in the yield of the extracts, with higher yields at the highest temperature. The oleuropein content in the extracts ranged from 80.5 to 82.9 mg g-1, and the extract presented different classes of compounds. The total phenolic components were in the range of 10.5 to 12.8 mg GAE g-1 of extract. The best condition to obtain an extract with the highest yield and total phenolic compounds, and suitable oleuropein content and antioxidant capacity was 60 °C, 10 MPa and 0.5 mL min-1.IntroductionThe olive tree (Olea europaea L.) is a fruit tree used for ornamental purposes and to produce olives. The production of the fruits is the main objective of its cultivation, aiming to obtain the olive oil as the final product. However, the olive leaves have essential components for human health and high polyphenol content ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1080/87559129.2010.496021", "ISBN" : "8755-9129", "ISSN" : "87559129", "abstract" : "Since phenolic compounds have been known as strong antioxidants, studies on olive leaves have attracted the investigators due to the richness of phenolic compounds in olive leaves. Recently, olive leaves are used in medicine, cosmetics, and in pharmaceutical products. It has a high potential for industrial exploitation in the food industry. In this study, the importance of olive leaves is briefly given, the composition of olive leaves, main phenolics in olive leaves and their health effects are described. Studies conducted on technological usage of olive leaves are reviewed. The future of olive leaves for the food industry is discussed. It is expected that this study will be beneficial to academic and industrial researchers interested in antioxidants, food additives, functional foods, and olive leaves.", "author" : [ { "dropping-particle" : "", "family" : "Erbay", "given" : "Zafer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Icier", "given" : "Filiz", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Food Reviews International", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2010" ] ] }, "page" : "319-334", "title" : "The importance and potential uses of olive leaves", "type" : "article-journal", "volume" : "26" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Erbay and Icier, 2010)", "plainTextFormattedCitation" : "(Erbay and Icier, 2010)", "previouslyFormattedCitation" : "(Erbay and Icier, 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Erbay and Icier, 2010).Oleuropein and its derivative, the hydroxytyrosol, are the predominant phenolic compounds in the olive leaves ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1080/87559129.2010.496021", "ISBN" : "8755-9129", "ISSN" : "87559129", "abstract" : "Since phenolic compounds have been known as strong antioxidants, studies on olive leaves have attracted the investigators due to the richness of phenolic compounds in olive leaves. Recently, olive leaves are used in medicine, cosmetics, and in pharmaceutical products. It has a high potential for industrial exploitation in the food industry. In this study, the importance of olive leaves is briefly given, the composition of olive leaves, main phenolics in olive leaves and their health effects are described. Studies conducted on technological usage of olive leaves are reviewed. The future of olive leaves for the food industry is discussed. It is expected that this study will be beneficial to academic and industrial researchers interested in antioxidants, food additives, functional foods, and olive leaves.", "author" : [ { "dropping-particle" : "", "family" : "Erbay", "given" : "Zafer", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Icier", "given" : "Filiz", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Food Reviews International", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2010" ] ] }, "page" : "319-334", "title" : "The importance and potential uses of olive leaves", "type" : "article-journal", "volume" : "26" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Erbay and Icier, 2010)", "plainTextFormattedCitation" : "(Erbay and Icier, 2010)", "previouslyFormattedCitation" : "(Erbay and Icier, 2010)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Erbay and Icier, 2010, Solarte-Toro et al., 2018). Oleuropein is a bitter-tasting glycoside which accounts for 73% of the total leaf phenolic constituents ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.jnutbio.2003.12.010", "ISBN" : "0955-2863", "ISSN" : "09552863", "PMID" : "15302080", "abstract" : "The potential protective effects of oleuropein, a dietary antioxidant of olive oil, has been investigated in the isolated rat heart. The organs were subjected to 30 minutes of no-flow global ischemia and then reperfused. At different time intervals, the coronary effluent was collected and assayed for creatine kinase activity as well as for reduced and oxidized glutathione. In addition, the extent of lipid peroxidation was evaluated by measuring thiobarbituric acid reactive substance concentration in cardiac muscle. Pretreatment with 20 \u03bcg/g oleuropein before ischemia resulted in a significant decrease in creatine kinase and reduced glutathione release in the perfusate. The protective effect of oleuropein against the post-ischemic oxidative burst was investigated by measuring the release, in the coronary effluent, of oxidized glutathione, a sensitive marker of heart's exposure to oxidative stress. Reflow in ischemic hearts was accompanied by a prompt release of oxidized glutathione; in ischemic hearts pretreated with oleuropein, this release was significantly reduced. Membrane lipid peroxidation was also prevented by oleuropein. The reported data provide the first experimental evidence of a direct cardioprotective effect of oleuropein in the acute events that follow coronary occlusion, likely because of its antioxidant properties. This finding strengthens the hypothesis that the nutritional benefit of olive oil in the prevention of coronary heart disease can be also related to the high content of oleuropein and its derivatives. Moreover, our data, together with the well documented antithrombotic and antiatherogenic activity of olive oil polyphenols, indicate these antioxidants as possible therapeutic tools for the pharmacological treatment of coronary heart disease as well as in the case of cardiac surgery, including transplantation. \u00a9 2004 Elsevier Inc. All rights reserved.", "author" : [ { "dropping-particle" : "", "family" : "Manna", "given" : "Caterina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Migliardi", "given" : "Valentina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Golino", "given" : "Paolo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Scognamiglio", "given" : "Annalisa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Galletti", "given" : "Patrizia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chiariello", "given" : "Massimo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zappia", "given" : "Vincenzo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Nutritional Biochemistry", "id" : "ITEM-1", "issue" : "8", "issued" : { "date-parts" : [ [ "2004" ] ] }, "page" : "461-466", "title" : "Oleuropein prevents oxidative myocardial injury induced by ischemia and reperfusion", "type" : "article-journal", "volume" : "15" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.3390/12051153", "ISBN" : "1420-3049", "ISSN" : "14203049", "PMID" : "17873849", "abstract" : "We report the determination of phenolic compounds in olive leaves by reversed-phase HPLC/DAD, and the evaluation of their in vitro activity against several microorganisms that may be causal agents of human intestinal and respiratory tract infections, namely Gram positive (Bacillus cereus, B. subtilis and Staphylococcus aureus), Gram negative bacteria (Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae) and fungi (Candida albicans and Cryptococcus neoformans). Seven phenolic compounds were identified and quantified: caffeic acid, verbascoside, oleuropein, luteolin 7-O-glucoside, rutin, apigenin 7-O-glucoside and luteolin 4\u2019-O-glucoside. At low concentrations olive leafs extracts showed an unusual combined antibacterial and antifungal action, which suggest their great potential as nutraceuticals, particulalry as a source of phenolic compounds.", "author" : [ { "dropping-particle" : "", "family" : "Pereira", "given" : "Ana Paula", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ferreira", "given" : "Isabel C.F.R.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Marcelino", "given" : "Filipa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Valent\u00e3o", "given" : "Patricia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Andrade", "given" : "Paula B.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Seabra", "given" : "Rosa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Estevinho", "given" : "Leticia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bento", "given" : "Albino", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pereira", "given" : "Jos\u00e9 Alberto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Molecules", "id" : "ITEM-2", "issue" : "5", "issued" : { "date-parts" : [ [ "2007" ] ] }, "page" : "1153-1162", "title" : "Phenolic compounds and antimicrobial activity of olive (Olea europaea L. Cv. Cobran\u00e7osa) leaves", "type" : "article-journal", "volume" : "12" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Manna et al., 2004; Pereira et al., 2007)", "plainTextFormattedCitation" : "(Manna et al., 2004; Pereira et al., 2007)", "previouslyFormattedCitation" : "(Manna et al., 2004; Pereira et al., 2007)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Pereira et al., 2007). It has high antioxidant and anti-inflammatory power ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.lfs.2005.07.029", "ISBN" : "0024-3205", "ISSN" : "00243205", "PMID" : "16236331", "abstract" : "Patients with diabetes mellitus are likely to develop certain complication such as retinopathy, nephropathy and neuropathy as a result of oxidative stress and overwhelming free radicals. Treatment of diabetic patients with antioxidant may be of advantage in attenuating these complications. Oleuropein, the active constituent of olive leaf (Olea europaea), has been endowed with many beneficial and health promoting properties mostly linked to its antioxidant activity. This study aimed to evaluate the significance of supplementation of oleuropein in reducing oxidative stress and hyperglycemia in alloxan-induced diabetic rabbits. After induction of diabetes, a significant rise in plasma and erythrocyte malondialdehyde (MDA) and blood glucose as well as alteration in enzymatic and non-enzymatic antioxidants was observed in all diabetic animals. During 16 weeks of treatment of diabetic rabbits with 20 mg/kg body weight of oleuropein the levels of MDA along with blood glucose and most of the enzymatic and non-enzymatic antioxidants were significantly restored to establish values that were not different from normal control rabbits. Untreated diabetic rabbits on the other hand demonstrated persistent alterations in the oxidative stress marker MDA, blood glucose and the antioxidant parameters. These results demonstrate that oleuropein may be of advantage in inhibiting hyperglycemia and oxidative stress induced by diabetes and suggest that administration of oleuropein may be helpful in the prevention of diabetic complications associated with oxidative stress. ?? 2005 Elsevier Inc. All rights reserved.", "author" : [ { "dropping-particle" : "", "family" : "Al-Azzawie", "given" : "Hasan Fayadh", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Alhamdani", "given" : "Mohamed Saiel Saeed", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Life Sciences", "id" : "ITEM-1", "issue" : "12", "issued" : { "date-parts" : [ [ "2006" ] ] }, "page" : "1371-1377", "title" : "Hypoglycemic and antioxidant effect of oleuropein in alloxan-diabetic rabbits", "type" : "article-journal", "volume" : "78" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1002/1438-9312(200210)104:9/10<677::AID-EJLT677>3.0.CO;2-M", "ISBN" : "1438-9312", "ISSN" : "14387697", "abstract" : "We have been studying the biological activities of olive oil phenols in vitro and in vivo including the bioavailability of phenols and their effects in animals and humans. In vitro, ortho-diphenolic (catecholic) compounds such as oleuropein and hydroxytyrosol exert potent antioxidant activities, such as inhibition of low density lipoproteins oxidation and free radical scavenging. They modify pathophysiological processes at cellular level favorably, e.g. by inhibiting not only the production of superoxide anions and the respiratory burst of neutrophils, but also platelet aggregation and the production of thromboxane and leukotriene B4 by neutrophils. Finally, oleuropein stimulates the release of nitric oxide by macrophages. Most important, recent data demonstrate that oleuropein and hydroxytyrosol, active at concentrations in the low \u03bcM range, are dose-dependently absorbed by humans and rats and, at very low doses, maintain their antioxidant activities in vivo. This is indicated by the increase in plasma antioxidant capacity and the reduced excretion of isoprostanes in humans and in sidestream smoke-exposed rats. To this end, our studies demonstrated that high quality, phenol-rich olive oil provides bioactive compounds that exert salubrious effects in humans and thus may contribute to the beneficial properties of the Mediterranean diet.", "author" : [ { "dropping-particle" : "", "family" : "Visioli", "given" : "Francesco", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Galli", "given" : "Claudio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Galli", "given" : "Giovanni", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Caruso", "given" : "Donatella", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European Journal of Lipid Science and Technology", "id" : "ITEM-2", "issue" : "9-10", "issued" : { "date-parts" : [ [ "2002" ] ] }, "page" : "677-684", "title" : "Biological activities and metabolic fate of olive oil phenols", "type" : "article-journal", "volume" : "104" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Al-Azzawie and Alhamdani, 2006; Visioli et al., 2002)", "plainTextFormattedCitation" : "(Al-Azzawie and Alhamdani, 2006; Visioli et al., 2002)", "previouslyFormattedCitation" : "(Al-Azzawie and Alhamdani, 2006; Visioli et al., 2002)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Al-Azzawie and Alhamdani, 2006), antimicrobial ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.copbio.2011.09.006", "ISBN" : "1879-0429 (Electronic)\r0958-1669 (Linking)", "ISSN" : "09581669", "PMID" : "22000808", "abstract" : "The Mediterranean diet is associated with a lower incidence of chronic degenerative diseases and higher life expectancy. These health benefits have been partially attributed to the dietary consumption of extra virgin olive oil (EVOO) by Mediterranean populations, and more specifically the phenolic compounds naturally present in EVOO. Studies involving humans and animals (in vivo and in vitro) have demonstrated that olive oil phenolic compounds have potentially beneficial biological effects resulting from their antimicrobial, antioxidant and anti-inflammatory activities. This paper summarizes current knowledge on the biological activities of specific olive oil phenolic compounds together with information on their concentration in EVOO, bioavailability and stability over time.", "author" : [ { "dropping-particle" : "", "family" : "Cicerale", "given" : "S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lucas", "given" : "LJ", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Keast", "given" : "RSJ", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Current Opinion in Biotechnology", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2012" ] ] }, "page" : "129-135", "publisher" : "Elsevier Ltd", "title" : "Antimicrobial, antioxidant and anti-inflammatory phenolic activities in extra virgin olive oil", "type" : "article-journal", "volume" : "23" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Cicerale et al., 2012)", "plainTextFormattedCitation" : "(Cicerale et al., 2012)", "previouslyFormattedCitation" : "(Cicerale et al., 2012)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Cicerale et al., 2012), antiviral ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.bbrc.2007.01.071", "ISBN" : "0006-291X (Print)\\r0006-291X (Linking)", "ISSN" : "0006291X", "PMID" : "17261269", "abstract" : "We have identified oleuropein (Ole) and hydroxytyrosol (HT) as a unique class of HIV-1 inhibitors from olive leaf extracts effective against viral fusion and integration. We used molecular docking simulation to study the interactions of Ole and HT with viral targets. We find that Ole and HT bind to the conserved hydrophobic pocket on the surface of the HIV-gp41 fusion domain by hydrogen bonds with Q577 and hydrophobic interactions with I573, G572, and L568 on the gp41 N-terminal heptad repeat peptide N36, interfering with formation of the gp41 fusion-active core. To test and confirm modeling predications, we examined the effect of Ole and HT on HIV-1 fusion complex formation using native polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Ole and HT exhibit dose-dependent inhibition on HIV-1 fusion core formation with EC50s of 66-58 nM, with no detectable toxicity. Our findings on effects of HIV-1 integrase are reported in the subsequent article. \u00a9 2007 Elsevier Inc. All rights reserved.", "author" : [ { "dropping-particle" : "", "family" : "Lee-Huang", "given" : "Sylvia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huang", "given" : "Philip Lin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zhang", "given" : "Dawei", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lee", "given" : "Jae Wook", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bao", "given" : "Ju", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sun", "given" : "Yongtao", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chang", "given" : "Young Tae", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zhang", "given" : "John", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huang", "given" : "Paul Lee", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Biochemical and Biophysical Research Communications", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2007" ] ] }, "page" : "872-878", "title" : "Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: Part I. Integrase inhibition", "type" : "article-journal", "volume" : "354" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Lee-Huang et al., 2007)", "plainTextFormattedCitation" : "(Lee-Huang et al., 2007)", "previouslyFormattedCitation" : "(Lee-Huang et al., 2007)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Lee-Huang et al., 2007), antitumor ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1007/s10616-009-9191-2", "ISBN" : "0920-9069 (Print)\\r0920-9069 (Linking)", "ISSN" : "09209069", "PMID" : "19353300", "abstract" : "Olive oil intake has been shown to induce significant levels of apoptosis in various cancer cells. These anti-cancer properties are thought to be mediated by phenolic compounds present in olive. These beneficial health effects of olive have been attributed, at least in part, to the presence of oleuropein and hydroxytyrosol. In this study, oleuropein and hydroxytyrosol, major phenolic compound of olive oil, was studied for its effects on growth in MCF-7 human breast cancer cells using assays for proliferation (MTT assay), cell viability (Guava ViaCount assay), cell apoptosis, cellcycle (flow cytometry). Oleuropein or hydroxytyrosol decreased cell viability, inhibited cell proliferation, and induced cell apoptosis in MCF-7 cells. Result of MTT assay showed that 200 mug/mL of oleuropein or 50 mug/mL of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein or hydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cell proliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited statistically significant block of G(1) to S phase transition manifested by the increase of cell number in G(0)/G(1) phase.", "author" : [ { "dropping-particle" : "", "family" : "Han", "given" : "Junkyu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Talorete", "given" : "Terence P N", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yamada", "given" : "Parida", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Isoda", "given" : "Hiroko", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cytotechnology", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2009" ] ] }, "page" : "45-53", "title" : "Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells", "type" : "article-journal", "volume" : "59" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1002/mnfr.200800204", "ISBN" : "1613-4125", "ISSN" : "16134125", "PMID" : "19194970", "abstract" : "Olive oil compounds is a dynamic research area because Mediterranean diet has been shown to protect against cardiovascular disease and cancer. Olive leaves, an easily available natural material of low cost, share possibly a similar wealth of health benefiting bioactive phytochemicals. In this work, we investigated the antioxidant potency and antiproliferative activity against cancer and endothelial cells of water and methanol olive leaves extracts and analyzed their content in phytochemicals using LC-MS and LC-UV-SPE-NMR hyphenated techniques. Olive-leaf crude extracts were found to inhibit cell proliferation of human breast adenocarcinoma (MCF-7), human urinary bladder carcinoma (T-24) and bovine brain capillary endothelial (BBCE). The dominant compound of the extracts was oleuropein; phenols and flavonoids were also identified. These phytochemicals demonstrated strong antioxidant potency and inhibited cancer and endothelial cell proliferation at low micromolar concentrations, which is significant considering their high abundance in fruits and vegetables. The antiproliferative activity of crude extracts and phytochemicals against the cell lines used in this study is demonstrated for the first time.", "author" : [ { "dropping-particle" : "", "family" : "Goulas", "given" : "Vlassios", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Exarchou", "given" : "Vassiliki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Troganis", "given" : "Anastassios N.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Psomiadou", "given" : "Eleni", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fotsis", "given" : "Theodoros", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Briasoulis", "given" : "Evangelos", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gerothanassis", "given" : "Ioannis P.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Molecular Nutrition and Food Research", "id" : "ITEM-2", "issue" : "5", "issued" : { "date-parts" : [ [ "2009" ] ] }, "page" : "600-608", "title" : "Phytochemicals in olive-leaf extracts and their antiproliferative activity against cancer and endothelial cells", "type" : "article-journal", "volume" : "53" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Goulas et al., 2009; Han et al., 2009)", "plainTextFormattedCitation" : "(Goulas et al., 2009; Han et al., 2009)", "previouslyFormattedCitation" : "(Goulas et al., 2009; Han et al., 2009)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Goulas et al., 2009), anticancer ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.3945/jn.109.104992", "ISBN" : "0022-3166", "ISSN" : "1541-6100", "PMID" : "19776181", "abstract" : "Chronic exposure to solar UV radiation damages skin, increasing its thickness and reducing its elasticity, and causes skin cancer. Our aim in this study was to examine the effects of an olive leaf extract and its component oleuropein on skin damage and the incidence of skin tumors caused by long-term UVB irradiation in hairless mice. Male hairless mice (5 wk old) were divided into 6 groups, including a non-UVB group, a vehicle-treated UVB group (control), 2 olive leaf extract-treated UVB groups, and 2 oleuropein-treated UVB groups. Five groups were UVB irradiated (36-180 mJ/cm(2)) 3 times each week for 30 wk and skin thickness and elasticity after UVB irradiation were measured every week. Olive leaf extract (300 and 1000 mg/kg) and oleuropein (10 and 25 mg/kg) were administered orally twice daily every day for 30 wk. The extract and oleuropein significantly inhibited increases in skin thickness and reductions in skin elasticity, and skin carcinogenesis and tumor growth. Furthermore, they prevented increases in the expression of matrix metalloproteinase (MMP)-2, MMP-9, and MMP-13 as well as in levels of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) in the skin. Based on histological evaluation, they prevented increases in the expression of Ki-67 and CD31-positive cells induced by the irradiation. These results suggest that the preventative effects of the olive leaf extract and oleuropein on chronic UVB-induced skin damage and carcinogenesis and tumor growth may be due to inhibition of the expression of VEGF, MMP-2, MMP-9, and MMP-13 through a reduction in COX-2 levels.", "author" : [ { "dropping-particle" : "", "family" : "Kimura", "given" : "Yoshiyuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sumiyoshi", "given" : "Maho", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "The Journal of nutrition", "id" : "ITEM-1", "issue" : "11", "issued" : { "date-parts" : [ [ "2009" ] ] }, "page" : "2079-86", "title" : "Olive leaf extract and its main component oleuropein prevent chronic ultraviolet B radiation-induced skin damage and carcinogenesis in hairless mice.", "type" : "article-journal", "volume" : "139" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Kimura and Sumiyoshi, 2009)", "plainTextFormattedCitation" : "(Kimura and Sumiyoshi, 2009)", "previouslyFormattedCitation" : "(Kimura and Sumiyoshi, 2009)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Kimura and Sumiyoshi, 2009), and neuroprotective ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.jsps.2010.05.005", "ISBN" : "1319-0164 (Print)\\r1319-0164 (Linking)", "ISSN" : "13190164", "PMID" : "23964170", "abstract" : "Traditional diets of people living in the Mediterranean basin are, among other components, very rich in extra-virgin olive oil, the most typical source of visible fat. Olive is a priceless source of monounsaturated and di-unsaturated fatty acids, polyphenolic antioxidants and vitamins. Oleuropein is the main glycoside in olives and is responsible for the bitter taste of immature and unprocessed olives. Chemically, oleuropein is the ester of elenolic acid and 3,4-dihydroxyphenyl ethanol, which possesses beneficial effects on human health, such as antioxidant, antiatherogenic, anti-cancer, anti-inflammatory and antimicrobial properties. The phenolic fraction extracted from the leaves of the olive tree, which contains significant amounts of oleuropein, prevents lipoprotein oxidation. In addition, oleuropein has shown cardioprotective effect against acute adriamycin cardiotoxicity and an anti-ischemic and hypolipidemic activities. Recently, oleuropein has shown neuroprotection by forming a non-covalent complex with the A\u03b2 peptide, which is a key hallmark of several degenerative diseases like Alzheimer and Parkinson. Thus, a large mass of research has been accumulating in the area of olive oil, in the attempt to provide evidence for the health benefits of olive oil consumption and to scientifically support the widespread adoption of traditional Mediterranean diet as a model of healthy eating. These results provide a molecular basis for some of the benefits potentially coming from oleuropein consumption and pave the way to further studies on the possible pharmacological use of oleuropein to prevent or to slow down the cardiovascular and neurodegenerative diseases. \u00a9 2010.", "author" : [ { "dropping-particle" : "", "family" : "Omar", "given" : "Syed Haris", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Saudi Pharmaceutical Journal", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2010" ] ] }, "page" : "111-121", "publisher" : "King Saud University", "title" : "Cardioprotective and neuroprotective roles of oleuropein in olive", "type" : "article-journal", "volume" : "18" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Omar, 2010a)", "plainTextFormattedCitation" : "(Omar, 2010a)", "previouslyFormattedCitation" : "(Omar, 2010a)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Omar, 2010a) actions, beneficial effects for cardiovascular diseases ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1186/s12967-014-0219-9", "ISBN" : "1479-5876 (Electronic)", "ISSN" : "1479-5876", "PMID" : "25086598", "abstract" : "The overall health beneficial action of olive oil phenolic components is well established. Recent studies have elucidated the biological effects of two isolated compounds, namely oleuropein and hydroxytyrosol, with particular attention on their antioxidant activity. Thus, a protective action has been demonstrated in preclinical studies against several diseases, especially cardiovascular and metabolic disorders. The present review will describe the biological effects of oleuropein and hydroxytyrosol, with particular attention on the molecular mechanism underlying the protective action on cardiovascular and metabolic alterations, as demonstrated by in vitro and in vivo experimental studies performed with the isolated compounds.", "author" : [ { "dropping-particle" : "", "family" : "Bulotta", "given" : "Stefania", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Celano", "given" : "Marilena", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lepore", "given" : "Saverio Massimo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Montalcini", "given" : "Tiziana", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pujia", "given" : "Arturo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Russo", "given" : "Diego", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Translational Medicine", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2014" ] ] }, "page" : "219", "title" : "Beneficial effects of the olive oil phenolic components oleuropein and hydroxytyrosol: focus on protection against cardiovascular and metabolic diseases", "type" : "article-journal", "volume" : "12" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Bulotta et al., 2014)", "plainTextFormattedCitation" : "(Bulotta et al., 2014)", "previouslyFormattedCitation" : "(Bulotta et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Bulotta et al., 2014), and other beneficial effects on human health.Pressurized liquid extraction (PLE) employs GRAS (Generally Recognized as Safe) liquid solvents at high pressures and operating temperature below the boiling point to achieve fast and efficient extraction (Albarelli et al., 2016). PLE generally has a higher percentage of mass yield compared to conventional extraction techniques for the extraction of bioactive products with high polarity ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/ac9508199", "author" : [ { "dropping-particle" : "", "family" : "Richter", "given" : "Bruce E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jones", "given" : "Brian A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ezzell", "given" : "John L", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Porter", "given" : "Nathan L", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Corporation", "given" : "Dionex", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Way", "given" : "Titan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Box", "given" : "P O", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-1", "issue" : "6", "issued" : { "date-parts" : [ [ "1996" ] ] }, "page" : "1033-1039", "title" : "Accelerated Solvent Extraction : A Technique for Sample Preparation", "type" : "article-journal", "volume" : "68" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.trac.2010.03.014", "author" : [ { "dropping-particle" : "", "family" : "Nieto", "given" : "Antonio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Borrull", "given" : "Francesc", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pocurull", "given" : "Eva", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Marce", "given" : "Rosa Maria", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-2", "issue" : "7", "issued" : { "date-parts" : [ [ "2010" ] ] }, "title" : "Pressurized liquid extraction : A useful technique to extract pharmaceuticals and personal-care products from sewage sludge", "type" : "article-journal", "volume" : "29" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Nieto et al., 2010; Richter et al., 1996)", "plainTextFormattedCitation" : "(Nieto et al., 2010; Richter et al., 1996)", "previouslyFormattedCitation" : "(Nieto et al., 2010; Richter et al., 1996)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Setyaningsih et al., 2016). Adjustments of the process parameters (temperature, pressure and solvent flow) may promote greater selectivity and increase the yield of the extracted compounds. These characteristics make PLE a clean alternative technology for the extraction of biocomposites of interest to the food, pharmaceutical and cosmetic industries.Therefore, it is possible to obtain oleuropein by employing several new techniques with promising results like ultrasound (Cabrera-Ba?egil et al., 2017) and extraction using homogenization with dimethyl sulfoxide (DMSO) and solid-phase extraction (Crawford et al., 2018). However, the PLE with pressurized ethanol and in a continuous flow has not been sufficiently explored. Thus, the main purpose of this study was to extract and characterize the phenolic compounds from olive leaves using pressurized ethanol in a continuous flow. The operating conditions evaluated were the temperature, pressure, and flow and the extracts were assessed for the contents of oleuropein, total phenolic compounds, and antioxidant activities. Furthermore, the phenolic compounds profile was also determined.Materials and Methods2.1 Raw materialThe raw material used was from commercial cultivation of olive trees located in the mountain range of Mantiqueira, S?o Paulo (22?00’48,6” S 46?37’59,4” W), Brazil. The olive varieties used were Arbequina, Koroneiki, and Arbosana. The leaves were separated manually from the branches and dried in a greenhouse with forced air circulation (Lab Store, New Ethics, model: 400/4ND, power: 1580 W) at 35 ?C for approximately 36 h (until constant weight) The solvent used for extraction was absolute ethyl alcohol 99.5 GL (Nuclear, S?o Paulo, Brazil) without previous treatment. Moisture, ash, lipid, protein, crude fiber, and carbohydrate contents were determined according to the Association of Official Analytical Chemists guidelinesADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "author" : [ { "dropping-particle" : "", "family" : "AOAC", "given" : "", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Washington, D.C. USA: A.O.A.C,", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2004" ] ] }, "title" : "Official methods of analysis of the Association of Official Analytical Chemists (19th ed.)", "type" : "webpage" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(AOAC, 2004)", "plainTextFormattedCitation" : "(AOAC, 2004)", "previouslyFormattedCitation" : "(AOAC, 2004)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }. All analyzes were performed in triplicates.2.2 Extraction with pressurized fluid The extractions were conducted using a 2? factorial design with triplicates at the central point. Table 1 shows the factors and levels used in the factorial design for extraction using pressurized ethanol. Table 1: Factors and levels of factorial design 23 with triplicates at the central point for the extraction from olive leaves using pressurized ethanolFactorsUnitsLevels(-1)(0)(1)Temperature°C304560PressureMPa101520Flow ratemL min-10.50.751.0Initially, 4 g of the dried and ground leaves were added to the extractor vessel. Then, the extraction system was filled with ethanol under the conditions of temperature, pressure and flow rate proposed in Table 1. The kinetic extraction was of 300 min for each condition. The obtained ethanolic extracts were evaporated in a circulating air oven at 55 ?C until constant weight. The mass yield (%) of the dry extracts was determined using an analytical balance (Denver Instrument, model: APX-200). 2.3 Analysis of oleuropein content in the extractsFor the analysis of the oleuropein content, extractions were made for all the experimental conditions described in Table 1. The ethanol was evaporated, and the extracts were analyzed. Analyzes were performed on High-Performance Liquid Chromatography (HPLC) using a C-18 column. The conditions for analysis were: mobile phase: methanol: water (50:50) using methanol with 1.0 μg μL-1 concentration, flow rate: 1 mL min-1, wavelength: 280 nm, oven temperature: 30 °C and injected volume: 5 μL. The oleuropein content was obtained using a five-point analytical curve of oleuropein (0.125–0.75 mg. mL-1). The analytical curve was linear (R2 = 0.985). The analysis was conducted in triplicate. 2.4 Determination of total phenolic components content and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assayThe Folin-Ciocalteu method was used to determine the total phenolic content present in the samples ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/jf048396b", "ISBN" : "0021-8561", "ISSN" : "00218561", "PMID" : "15740008", "abstract" : "Polyphenols, widely spread in our diet by the consumption of plant food products, are commonly determined using Folin-Ciocalteu reagent that interacts with other different reducing nonphenolic substances and leads to an overestimation of polyphenol content. In this paper we report an optimized Folin-Ciocalteu method to specifically determine the contents of total polyphenols and vitamin C. After the optimal conditions for the colorimetric assay were set, solid-phase extraction (Oasis HLB (hydrophilic-lipophilic balance)) was carried out to eliminate the water-soluble reducing interferences including vitamin C. Colorimetric correction was thus performed by subtracting interfering substances contained in the water washing extract from the raw extract. Moreover, vitamin C present in the water washing extract can be destroyed by heating and thus colorimetrically deduced. This procedure was set up with synthetic solutions and validated on different extracts from fruit products.", "author" : [ { "dropping-particle" : "", "family" : "Georg\u00e9", "given" : "Stephane", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Brat", "given" : "Pierre", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Alter", "given" : "Pascaline", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Amiot", "given" : "Marie J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Agricultural and Food Chemistry", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2005" ] ] }, "page" : "1370-1373", "title" : "Rapid determination of polyphenols and vitamin C in plant-derived products", "type" : "article-journal", "volume" : "53" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Georg\u00e9 et al., 2005)", "plainTextFormattedCitation" : "(Georg\u00e9 et al., 2005)", "previouslyFormattedCitation" : "(Georg\u00e9 et al., 2005)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Georgé et al., 2005). The content of phenolic compounds was expressed in milligrams of gallic acid equivalents per gram of extract (mg GAE g-1 de extract). The antioxidant activity of the extracts was performed using the free radical scavenging method DPPH, according to the methodology proposed by ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/jf049320i", "ISBN" : "0021-8561", "ISSN" : "00218561", "PMID" : "15796575", "abstract" : "Phenolic compounds from soft and hard wheat and their milling fractions were extracted into distilled deionized water, and their in vitro antioxidant activities were evaluated. Wheat samples were used as such (nontreated) or subjected to pH adjustment (treated) in order to simulate gastrointestinal pH conditions. The total phenolic content (TPC) was determined using Folin-Ciocalteu's procedure. The total antioxidant activity (TAA) was determined using Trolox equivalent antioxidant capacity assay and expressed as Trolox equivalents. The antioxidant activity of wheat extracts was also evaluated using the beta-carotene bleaching assay, scavenging of 2,2-diphenyl-1-picrylhydrazyl radical, and inhibition of oxidation of human low density lipoprotein cholesterol. The TPC, TAA, and antioxidant potential, evaluated using different methods of wheat samples, were significantly increased following gastrointestinal tract-simulated pH changes. Thus, digestion taking place in the gastrointestinal tract in vivo may also enhance the antioxidant properties of the extracts.", "author" : [ { "dropping-particle" : "", "family" : "Liyana-Pathirana", "given" : "Chandrika M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shahidi", "given" : "Fereidoon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Agricultural and Food Chemistry", "id" : "ITEM-1", "issue" : "7", "issued" : { "date-parts" : [ [ "2005" ] ] }, "page" : "2433-2440", "title" : "Antioxidant activity of commercial soft and hard wheat (Triticum aestivum L.) as affected by gastric pH conditions", "type" : "article-journal", "volume" : "53" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Liyana-Pathirana and Shahidi, 2005)", "plainTextFormattedCitation" : "(Liyana-Pathirana and Shahidi, 2005)", "previouslyFormattedCitation" : "(Liyana-Pathirana and Shahidi, 2005)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }Liyana-Pathirana and Shahidi, (2005). The concentration of the sample with a 50% reduction in DPPH (IC50) was calculated from the graph equation, consisting in the percentage of antioxidant activity versus concentration in μg mL-1.2.5 Characterization of the extracts by HPLC–DAD–MSn AnalysisThe extract of olive leaves obtained under conditions of 60 °C, 10 MPa and flow rate of 0.5 mL.min-1, which was the condition with the best result in the factorial design, was suspended in a methanol/formic acid mixture (99.5: 0.5 (v / v)) (8 mg of extract mL-1) and centrifuged at 3000 g for 5 min at 4 °C. The supernatant was filtered through 0.22 μm membranes (Millipore), and 5 μL was injected into the HPLC-DAD-MSn apparatus.A Shimadzu HPLC apparatus equipped with quaternary pumps (LC-20AD), a degasser (DGU-20A3R), and an injection valve (SIL-20A HT) with a 20 μL loop, connected in series to a DAD detector (SPD-M20A) and a mass spectrometer with an ion trap analyzer and an electrospray ionization (ESI) source (model AmaZon Speed, Bruker Daltonics) was used to assess the phenolic compounds. The phenolic compounds were separated on a C18 Synergi HydroRP column (4 μm, 250 mm × 4.6 mm, Phenomenex) at a flow rate of 0.7 mL.min-1 and a column temperature of 30 °C, using a mobile phase consisting of mixtures of water/formic acid [99.5:0.5 (v/v)] (solvent A) and methanol/formic acid [99.5:0.5 (v/v)] (solvent B) in an isocratic gradient from 40:60 (v/v) A/B for 30 min. The column eluate was split to allow only around 0.15 mL.min-1 to enter the ESI interface. The UV–VIS spectra were obtained between 200 and 600 nm, and the chromatograms were processed at 280 nm. The mass spectra were acquired with a scan range from m/z 100 to 800. The MS parameters were set as follows: ESI source operating in negative ion mode; dry gas (N2) temperature of 310 °C; flow rate of 8 L min-1 and nebulizer gas with 30 psi. MS2 and MS3 were set in manual mode applying fragmentation energy of 1.6 V. The phenolic compounds were identified based on the following information: elution order and retention time in the reversed phase column, UV–VIS and MS spectra features as compared to standards analyzed under the same conditions, and data available in the literature. Results and Discussion3.1 Physicochemical characterization of the raw materialTable 2 shows the moisture, ash, lipid, protein, crude fiber, and carbohydrate contents of the dried olive leaves obtained in the present study and those found in the literature.Table 2: Physicochemical characterization of the olive leaves used in the experiments (in dry mass). Component (%)Present studyLiterature*Moisture5.8 ± 0.054.8Ash5.6 ± 0.211.9 Lipids9.0 ± 0.73.7Protein13.0 ± 0.510.8Crude fiber3.4 ± 0.314.5Carbohydrate63.1 59.0The physicochemical characterization of the olive leaves demonstrates the similarity between the raw material used in this study and the one used in other studies available in the literature* ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "author" : [ { "dropping-particle" : "", "family" : "Coppa", "given" : "Carolina Fernanda Sengling Cebin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cebin", "given" : "Sengling", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rosim", "given" : "Roice Eliana", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Augusto", "given" : "Carlos", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "De", "family" : "Oliveira", "given" : "Fernandes", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Elisabete", "given" : "Christianne", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gon\u00e7alves", "given" : "Cintia Bernardo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Brazilian Jornal of Food Technology", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2017" ] ] }, "title" : "Extra\u00e7\u00e3o de oleurope\u00edna a partir de folhas de oliveira utilizando solvente hidroalco\u00f3lico Extraction of oleuropein from olive leaves using a hydroalcoholic solvent", "type" : "article-journal" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1590/0103-8478cr20131139", "abstract" : "The olive tree (Olea europaea L.) is a plant characteristic of the Mediterranean region that is cultivated in various countries of the world due to the great interest in the production of olives and their oil, which is rich in essential fatty acids. The leaves of the olive tree are an important by-product generated by pruning of trees that have signifi cant amount of phenolic and fatty acids important to health. Given the importance of compounds present in olive leaves and the recent crop in southern Brazil, this study aimed to perform an exploratory study on the proximate composition, total phenolic compounds, fatty acids and minerals present in the leaves of the \u2018Arbequina\u2019 variety from Ca\u00e7apava do Sul, RS, Brazil. The results showed that the leaves had 8.14\u00b10.24% of total lipids, with an amount of polyunsaturated fatty acids more than half of the total content of fatty acids, in special linolenic acid (18:3n3, 46,7%), which is present in the olive oil in general close to the concentration of 1%. The concentration of total phenolics was 30.21\u00b10.31mg GAE g-1 and the predominant minerals in the leaves were Ca, K, Mg, P and S (between 1153 and 11448mg g-1). The sodium concentration was considered low (65.4\u00b15.20mg g-1). These results demonstrate that the leaves from olive trees cultivated in the region of Ca\u00e7apava do Sul have great potential for use in dietary supplementation both human and animal because are rich in nutrients", "author" : [ { "dropping-particle" : "", "family" : "Cavalheiro", "given" : "Caroline Viegas", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rosso", "given" : "Vandrisa Dellinghausen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Paulus", "given" : "Eloi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cichoski", "given" : "Alexandre Jos\u00e9", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wagner", "given" : "Roger", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cristiano", "given" : "Ragagnin de Menezes", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barin", "given" : "Juliano Smanioto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Ci\u00eancia Rural", "id" : "ITEM-2", "issue" : "10", "issued" : { "date-parts" : [ [ "2014" ] ] }, "page" : "1874-1879", "title" : "Chemical composition of olive leaves (Olea europaea L.) from the region of Ca\u00e7apava do Sul, RS, Brazil", "type" : "article-journal", "volume" : "44" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Cavalheiro et al., 2014; Coppa et al., 2017)", "plainTextFormattedCitation" : "(Cavalheiro et al., 2014; Coppa et al., 2017)", "previouslyFormattedCitation" : "(Cavalheiro et al., 2014; Coppa et al., 2017)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Cavalheiro et al., 2014; Coppa et al., 2017). The moisture content directly influences the extraction process by limiting solvent penetration into the matrix pores and reducing mass transfer. Thus, reduced values of moisture content are essential. Therefore, it is advised to use a dry material in the extraction with pressurized liquid ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1007/978-88-470-5738-8", "ISBN" : "978-88-470-5737-1", "author" : [ { "dropping-particle" : "", "family" : "Moret", "given" : "Sabrina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Conte", "given" : "Lanfranco S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "chapter-number" : "3", "container-title" : "Il campione per l\u2019analisi chimica", "edition" : "1", "editor" : [ { "dropping-particle" : "", "family" : "Moret", "given" : "Sabrina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Purcato", "given" : "Giorgia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Conte", "given" : "Lanfranco S.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2014" ] ] }, "page" : "177-214", "publisher" : "Springer-Verlag Mailand", "title" : "Pressurized liquid extraction (PLE)", "type" : "chapter" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Moret and Conte, 2014)", "plainTextFormattedCitation" : "(Moret and Conte, 2014)", "previouslyFormattedCitation" : "(Moret and Conte, 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Moret and Conte, 2014).The olive leaves from cultivars such as Arbequina that are cultivated in the south of Brazil have an interesting chemical composition compared to varieties cultivated in other countries, due to their high protein content. The contents of ash (4.4%), proteins (12.2%) and lipids (8.1%) obtained by ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1590/0103-8478cr20131139", "abstract" : "The olive tree (Olea europaea L.) is a plant characteristic of the Mediterranean region that is cultivated in various countries of the world due to the great interest in the production of olives and their oil, which is rich in essential fatty acids. The leaves of the olive tree are an important by-product generated by pruning of trees that have signifi cant amount of phenolic and fatty acids important to health. Given the importance of compounds present in olive leaves and the recent crop in southern Brazil, this study aimed to perform an exploratory study on the proximate composition, total phenolic compounds, fatty acids and minerals present in the leaves of the \u2018Arbequina\u2019 variety from Ca\u00e7apava do Sul, RS, Brazil. The results showed that the leaves had 8.14\u00b10.24% of total lipids, with an amount of polyunsaturated fatty acids more than half of the total content of fatty acids, in special linolenic acid (18:3n3, 46,7%), which is present in the olive oil in general close to the concentration of 1%. The concentration of total phenolics was 30.21\u00b10.31mg GAE g-1 and the predominant minerals in the leaves were Ca, K, Mg, P and S (between 1153 and 11448mg g-1). The sodium concentration was considered low (65.4\u00b15.20mg g-1). These results demonstrate that the leaves from olive trees cultivated in the region of Ca\u00e7apava do Sul have great potential for use in dietary supplementation both human and animal because are rich in nutrients", "author" : [ { "dropping-particle" : "", "family" : "Cavalheiro", "given" : "Caroline Viegas", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rosso", "given" : "Vandrisa Dellinghausen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Paulus", "given" : "Eloi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cichoski", "given" : "Alexandre Jos\u00e9", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wagner", "given" : "Roger", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cristiano", "given" : "Ragagnin de Menezes", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barin", "given" : "Juliano Smanioto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Ci\u00eancia Rural", "id" : "ITEM-1", "issue" : "10", "issued" : { "date-parts" : [ [ "2014" ] ] }, "page" : "1874-1879", "title" : "Chemical composition of olive leaves (Olea europaea L.) from the region of Ca\u00e7apava do Sul, RS, Brazil", "type" : "article-journal", "volume" : "44" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Cavalheiro et al., 2014)", "plainTextFormattedCitation" : "(Cavalheiro et al., 2014)", "previouslyFormattedCitation" : "(Cavalheiro et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Cavalheiro et al., 2014) when evaluating olive leaves produced in Brazil, in the region of Ca?apava do Sul, RS, were similar to the results obtained in this study. 3.2 Extraction with pressurized liquidThe experimental design defined to obtain the kinetic extraction curves is shown in Table 3. It is observed that the highest extraction occurred up to approximately 70 min, after this period, the diffusion is predominant. The values of mass percentage yields ranged from 7.2 to 15.1% and are compatible with the yield values obtained by several authors. The temperature is the operational parameter with the highest positive effect in the extraction, providing a higher yield of the extract (14.5 to 15.1%) in the experiments with higher temperatures (60 oC), independently of the pressure and solvent flow rate. In this condition, it can be demonstrated that it is possible to use the lowest solvent flow rate and pressure aiming solvent and energy saving. This can be explained by the fact that temperature is a parameter of significant influence in a PLE process, being able to improve the extraction efficiency by disturbing the dipole and hydrogen bond interactions between the analyte and the matrix, decreasing the activation energy required for the desorption, and decreasing the surface tension and viscosity of the solvent ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/ac9508199", "author" : [ { "dropping-particle" : "", "family" : "Richter", "given" : "Bruce E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jones", "given" : "Brian A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ezzell", "given" : "John L", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Porter", "given" : "Nathan L", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Corporation", "given" : "Dionex", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Way", "given" : "Titan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Box", "given" : "P O", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-1", "issue" : "6", "issued" : { "date-parts" : [ [ "1996" ] ] }, "page" : "1033-1039", "title" : "Accelerated Solvent Extraction : A Technique for Sample Preparation", "type" : "article-journal", "volume" : "68" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1080/10406630701462916", "ISSN" : "10406638", "author" : [ { "dropping-particle" : "", "family" : "Moreno", "given" : "Erick", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Reza", "given" : "Joel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Trejo", "given" : "Arturo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Polycyclic Aromatic Compounds", "id" : "ITEM-2", "issue" : "4", "issued" : { "date-parts" : [ [ "2007" ] ] }, "page" : "239-260", "title" : "Extraction of polycyclic aromatic hydrocarbons from soil using water under subcritical conditions", "type" : "article-journal", "volume" : "27" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Moreno et al., 2007; Richter et al., 1996)", "plainTextFormattedCitation" : "(Moreno et al., 2007; Richter et al., 1996)", "previouslyFormattedCitation" : "(Moreno et al., 2007; Richter et al., 1996)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Moreno et al., 2007). In fact, the statistical analysis confirmed the significant influence of the temperature (p ≤ 0.05). The pressure did not present a statistically significant effect (p > 0.05). Generally, high pressure values allow the solvent to remain in the liquid form, even at temperatures above the boiling point and facilitate solvent penetration into the sample pores.In the case of the present study, the temperatures used (30-60 oC) were below the boiling point of the ethanol (78 oC), with no significant influence of the pressure on the yield. The flow range used was not statistically significant to change the mass percentage yield (p > 0.05). The flow is directly related to mass transfer mechanisms and should be evaluated whenever possible ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1007/978-88-470-5738-8", "ISBN" : "978-88-470-5737-1", "author" : [ { "dropping-particle" : "", "family" : "Moret", "given" : "Sabrina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Conte", "given" : "Lanfranco S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "chapter-number" : "3", "container-title" : "Il campione per l\u2019analisi chimica", "edition" : "1", "editor" : [ { "dropping-particle" : "", "family" : "Moret", "given" : "Sabrina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Purcato", "given" : "Giorgia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Conte", "given" : "Lanfranco S.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2014" ] ] }, "page" : "177-214", "publisher" : "Springer-Verlag Mailand", "title" : "Pressurized liquid extraction (PLE)", "type" : "chapter" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Moret and Conte, 2014)", "plainTextFormattedCitation" : "(Moret and Conte, 2014)", "previouslyFormattedCitation" : "(Moret and Conte, 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Moret and Conte, 2014). The significant effect of the curvature indicates that the quadratic terms of the independent parameters are essential. However, the linear regression model applied for the independent variables (Eq. 1) resulted in a linear correlation coefficient of 0.9959 and variance of 97.98% for the variable yield of olive leaf extract.RExt=11.46+3.33T R2=0.9959 (1)Where RExt is the extract yield (%), and T is the temperature (°C).Oleuropein yield values in the extracts were similar for all extraction conditions conducted (Table 3). Therefore, the effects of temperature, pressure, and ethanol flow were not statistically significant for the oleuropein content in the extracts (p> 0.05). Table 3: Yield of the extracts and oleuropein, phenolic compounds contents and antioxidant activity of olive leaf extracts obtained in the extraction with pressurized ethanol according to experimental design.ExpT (?C)P (MPa)F (mL min-1)YG (w/w) %YOLE (mg g-1 extract)Total phenolics (mg GAE g-1 extract)IC50 (μg mL-1)130100.57.986.610.5 ± 0.4 c36.1 ± 2.6 ab230200.59.786.811.7 ± 0.4 abc36.3 ± 1.6 ab330101.07.285.211.3 ± 0.4 abc32.2 ± 8.2 b430201.07.786.911.2 ± 0.3 bc29.3 ± 2.6 b545150.759.7 ± 0.585.5 ± 0.211.8 ± 0.4 abc46.4 ± 1.3 a660100.515.189.212.8 ± 0.5a42.4 ± 2.2 a760200.514.984.911.5 ± 0.4 abc43.5 ± 0.7 a860101.014.784.612.5 ± 0.6 a42.2 ± 0.4 a960201.014.580.512.1 ± 0.2 ab43.5 ± 0.6 aT is temperature, P is pressure, F is flow rate, YG is extract yield and YOLE is oleuropein yield. IC50: Concentration of extract required to decrease the concentration of DPPH by 50%. Different letters in the same column differ from each other by the Tukey test at 5% significance.3.4 Determination of total phenolic components and antioxidant activityTable 3 shows the total phenolic component contents and the antioxidant activity of the extracts. The values of the total phenolic components obtained ranged from 10.5 ± 0.4 to 12.8 ± 0.5 mg GAE g-1 of extract. The highest total phenolic compounds contents (11.5-12.8 mg GAE g-1 of extract) were observed in the extracts obtained using the highest temperature (60 oC). The increase in the pressure and flow rate did not result in a significant impact (p > 0.05) on the total phenolic content. Therefore, to obtain an extract with higher total phenolic compounds, it is recommended the experiment 6 (60 oC, 10 MPa and 0.5 mL min-1).It is observed that there is a variation of the results found in the literature regarding the phenolics in leaves of O. europaea, with some of them close to the results found in this study. The variations may occur according to the cultivar, harvest season, climate and olive growing conditions ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "author" : [ { "dropping-particle" : "", "family" : "Coppa", "given" : "Carolina Fernanda Sengling Cebin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cebin", "given" : "Sengling", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rosim", "given" : "Roice Eliana", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Augusto", "given" : "Carlos", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "De", "family" : "Oliveira", "given" : "Fernandes", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Elisabete", "given" : "Christianne", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gon\u00e7alves", "given" : "Cintia Bernardo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Brazilian Jornal of Food Technology", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2017" ] ] }, "title" : "Extra\u00e7\u00e3o de oleurope\u00edna a partir de folhas de oliveira utilizando solvente hidroalco\u00f3lico Extraction of oleuropein from olive leaves using a hydroalcoholic solvent", "type" : "article-journal" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Coppa et al., 2017)", "plainTextFormattedCitation" : "(Coppa et al., 2017)", "previouslyFormattedCitation" : "(Coppa et al., 2017)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Coppa et al., 2017).The antioxidant capacity results, expressed as IC50, are shown in Table 3. The values obtained demonstrate the good antioxidant capacity of the extracts, varying from 29.3 to 45.2 μg mL-1. The highest antioxidant capacity was observed in the extracts obtained at the lowest temperature (30 oC) (p ≤ 0.05), independently of the pressure and flow rates (p > 0.05). Therefore, the antioxidant activity of the extracts obtained was not correlated with the total phenolic content of the olive leaf extracts. Probably, the antioxidant behavior of the extracts is defined by the interactions between the various constituents and not only by the oleuropein content ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1007/s00216-008-2353-9", "ISBN" : "1618-2642", "ISSN" : "16182642", "PMID" : "18762919", "abstract" : "An experimental setup based on a 2(3) full-factorial, central-composite design was implemented with the aim of optimising the recovery of polyphenols from olive leaves by employing reusable and nontoxic solutions composed of water/ethanol/citric acid as extracting media. The factors considered were (i) the pH of the medium, (ii) the extraction time and (iii) the ethanol concentration. The model obtained produced a satisfactory fit to the data with regard to total polyphenol extraction (R(2) = 0.91, p = 0.0139), but not for the antiradical activity of the extracts (R(2) = 0.67, p = 0.3734). The second-order polynomial equation obtained after analysing the experimental data indicated that ethanol concentration and time mostly affected the extraction yield, but that increased pH values were unfavourable in this regard. The maximum theoretical yield was calculated to be 250.2 +/- 76.8 mg gallic acid equivalent per g of dry, chlorophyll-free tissue under optimal conditions (60% EtOH, pH 2 and 5 h). Liquid chromatography-electrospray ionisation mass spectrometry of the optimally obtained extract revealed that the principal phytochemicals recovered were luteolin 7-O-glucoside, apigenin 7-O-rutinoside and oleuropein, accompanied by smaller amounts of luteolin 3',7-O-diglucoside, quercetin 3-O-rutinoside (rutin), luteolin 7-O-rutinoside and luteolin 3'-O-glucoside. Simple linear regression analysis between the total polyphenol and antiradical activity values gave a low and statistically insignificant correlation (R(2) = 0.273, p > 0.05), suggesting that it is not the sheer amount of polyphenols that provides high antioxidant potency; instead, this potency is probably achieved through interactions among the various phenolic constituents.", "author" : [ { "dropping-particle" : "", "family" : "Mylonaki", "given" : "Stefania", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kiassos", "given" : "Elias", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Makris", "given" : "Dimitris P.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kefalas", "given" : "Panagiotis", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical and Bioanalytical Chemistry", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2008" ] ] }, "page" : "977-985", "title" : "Optimisation of the extraction of olive (Olea europaea) leaf phenolics using water/ethanol-based solvent systems and response surface methodology", "type" : "article-journal", "volume" : "392" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Mylonaki et al., 2008)", "plainTextFormattedCitation" : "(Mylonaki et al., 2008)", "previouslyFormattedCitation" : "(Mylonaki et al., 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Mylonaki et al., 2008). From the statistical analysis, it was verified that the temperature is the most critical factor in the extraction of these components, with prominence for antioxidant activity. In the operational condition of extraction of 30 ?C it was possible to obtain the highest antioxidant activity in the extracts (29.3 to 36.3 μg mL-1). The linear regression model (Eq. 3) obtained by the statistical analysis of the process variables presented a linear regression coefficient of 0.9739 and a model adjustment of 86.95%.DPPH IC50=38,19+4,72T (3)where DPPH IC50 is the antioxidant activity of extracts (μg mL-1), and T is the temperature.The use of olive leaf extract rich in oleuropein and other phenolic compounds with antioxidant activity is promising for the enrichment of olive oil, for example, increasing its polyphenol composition and its oxidative stability ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "author" : [ { "dropping-particle" : "", "family" : "Coppa", "given" : "Carolina Fernanda Sengling Cebin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cebin", "given" : "Sengling", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rosim", "given" : "Roice Eliana", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Augusto", "given" : "Carlos", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "De", "family" : "Oliveira", "given" : "Fernandes", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Elisabete", "given" : "Christianne", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gon\u00e7alves", "given" : "Cintia Bernardo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Brazilian Jornal of Food Technology", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2017" ] ] }, "title" : "Extra\u00e7\u00e3o de oleurope\u00edna a partir de folhas de oliveira utilizando solvente hidroalco\u00f3lico Extraction of oleuropein from olive leaves using a hydroalcoholic solvent", "type" : "article-journal" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1002/ejlt.201000445", "ISBN" : "5602222790", "ISSN" : "14387697", "abstract" : "The effect of the addition of olive leaf (Olea europaea, cv. Arbequina) extracts, i.e. hydroalcoholic (ethanol\u2013water 1:1; OHE), juice (OJ) and supercritical fluid-CO2 (OSFE) on the oxidative stability of vegetable oils with different unsaturation, such as soybean oil (SBO), canola oil (CO) and high oleic sunflower oil (HOSO), were studied at two concentrations (250 and 630 mg/kg oil, expressed as caffeic acid equivalent (CAE)). The extracts were characterized by the total phenolic content (Folin\u2013Ciocalteau method), phenol chromatographic profiles (LC-MS) and antioxidant activity (DPPH). OHE showed the highest phenol content (7.7 mg CAE/mL) while OJ and OSFE showed values of 5.4 and 2.2 mg CAE/mL, respectively. Oleuropein and its derivatives were the major phenolic compounds identified in OHE. The addition of 630 mg CAE/kg oil of OHE and OSFE to HOSO, SBO and CO showed an antioxidant effect, increasing significantly the induction time (IT) (p<0.05). That effect was highest when the system was moremonounsaturated. In contrast, OJ showed a pro-oxidant effect for all oils systems for both concentration studied. This behaviour could be attributed to the diphenol oxidase (PPO) activity.", "author" : [ { "dropping-particle" : "", "family" : "Jimenez", "given" : "Paula", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Masson", "given" : "Lilia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barriga", "given" : "Andr\u00e9s", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ch\u00e1vez", "given" : "Jorge", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Robert", "given" : "Paz", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European Journal of Lipid Science and Technology", "id" : "ITEM-2", "issue" : "4", "issued" : { "date-parts" : [ [ "2011" ] ] }, "page" : "497-505", "title" : "Oxidative stability of oils containing olive leaf extracts obtained by pressure, supercritical and solvent-extraction", "type" : "article-journal", "volume" : "113" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Coppa et al., 2017; Jimenez et al., 2011)", "plainTextFormattedCitation" : "(Coppa et al., 2017; Jimenez et al., 2011)", "previouslyFormattedCitation" : "(Coppa et al., 2017; Jimenez et al., 2011)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Jimenez et al., 2011; Coppa et al., 2017). 3.3 Characterization of the Extract by HPLC–DAD–MSn AnalysisThe HPLC–DAD chromatogram processed at 280 nm shows the separation of 18 peaks, corresponding to 21 compounds from olive leaf extracts (Figure 1). Considering that a description of phenolic identification from olive leaves had already been reported by ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/jf302896m", "ISBN" : "0021-8561", "ISSN" : "00218561", "PMID" : "23106267", "abstract" : "The reuse of agronomical residues is a pending goal for sustainable agriculture. Particular residues in olive-oil-producing countries are leaves, wastewater, and olive pomace. Olive leaves and wastewaters have been previously characterized by isolation of the phenolic fraction. However, olive pomace has not yet been qualitatively characterized as a source of phenols. Olive pomace extracts were obtained using superheated solvent extraction using 50:50 (v/v) water/ethanol as a leaching mixture at 160 \u00b0C. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry using a quadrupole-quadrupole-time-of-flight (QqTOF) hybrid mass analyzer (R = 25 000-45 000). Qualitative analysis was supported upon measurement of accurate masses for precursor and product ions as well as their isotopic distribution. Identification was focused on the main families of phenolic compounds present in extra virgin olive oil. The potential of this residue as a rich source of phenols with antioxidant properties has been proven. \u00a9 2012 American Chemical Society.", "author" : [ { "dropping-particle" : "", "family" : "Peralbo-Molina", "given" : "\u00c1ngela", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Priego-Capote", "given" : "Feliciano", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Luque De Castro", "given" : "Mar\u00eda Dolores", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Agricultural and Food Chemistry", "id" : "ITEM-1", "issue" : "46", "issued" : { "date-parts" : [ [ "2012" ] ] }, "page" : "11542-11550", "title" : "Tentative identification of phenolic compounds in olive pomace extracts using liquid chromatography-tandem mass spectrometry with a quadrupole- quadrupole-time-of-flight mass detector", "type" : "article-journal", "volume" : "60" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Peralbo-Molina et al., 2012)", "manualFormatting" : "Peralbo-Molina et al. (2012)", "plainTextFormattedCitation" : "(Peralbo-Molina et al., 2012)", "previouslyFormattedCitation" : "(Peralbo-Molina et al., 2012)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }Peralbo-Molina et al. (2012), ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1002/pca.2401", "ISBN" : "1099-1565", "ISSN" : "09580344", "PMID" : "22987739", "abstract" : "INTRODUCTION: Olea europaea L. leaves may be considered a cheap, easily available natural source of phenolic compounds. In a previous study we evaluated the possibility of obtaining bioactive phenolic compounds from olive leaves by pressurised liquid extraction (PLE) for their use as natural anti-oxidants. The alimentary use of these kinds of extract makes comprehensive knowledge of their composition essential.\\n\\nOBJECTIVE: To undertake a comprehensive characterisation of two olive-leaf extracts obtained by PLE using high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS).\\n\\nMETHOD: Olive leaves were extracted by PLE using ethanol and water as extraction solvents at 150\u00b0C and 200\u00b0C respectively. Separation was carried out in a HPLC system equipped with a C\u2081\u2088-column working in a gradient elution programme coupled to ESI-QTOF-MS operating in negative ion mode.\\n\\nRESULTS: This analytical platform was able to detect 48 compounds and tentatively identify 31 different phenolic compounds in these extracts, including secoiridoids, simple phenols, flavonoids, cinnamic-acid derivatives and benzoic acids. Lucidumoside C was also identified for the first time in olive leaves.\\n\\nCONCLUSION: The coupling of HPLC-ESI-QTOF-MS led to the in-depth characterisation of the olive-leaf extracts on the basis of mass accuracy, true isotopic pattern and tandem mass spectrometry (MS/MS) spectra. We may conclude therefore that this analytical tool is very valuable in the study of phenolic compounds in plant matrices.", "author" : [ { "dropping-particle" : "", "family" : "Quirantes-Pin\u00e9", "given" : "Rosa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lozano-S\u00e1nchez", "given" : "Jes\u00fas", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Herrero", "given" : "Miguel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ib\u00e1\u00f1ez", "given" : "Elena", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Segura-Carretero", "given" : "Antonio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fern\u00e1ndez-Guti\u00e9rrez", "given" : "Alberto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Phytochemical Analysis", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2013" ] ] }, "page" : "213-223", "title" : "HPLC-ESI-QTOF-MS as a powerful analytical tool for characterising phenolic compounds in olive-leaf extracts", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Quirantes-Pin\u00e9 et al., 2013)", "manualFormatting" : "Quirantes-Pin\u00e9 et al. (2013)", "plainTextFormattedCitation" : "(Quirantes-Pin\u00e9 et al., 2013)", "previouslyFormattedCitation" : "(Quirantes-Pin\u00e9 et al., 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }Quirantes-Piné et al. (2013), ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1080/00032719.2014.888732", "ISSN" : "1532236X", "abstract" : "High performance liquid chromatography coupled with electrospray ionization quadrupole-time-of-flight tandem mass spectrometry was used to profile the phenylethanoid glycosides in Lagotis brevituba. A total of twenty-three phenylethanoid glycosides were characterized by comparing the retention time and fragmentation with standards, accurate mass measurements, and fragmentation at low and high collision energies. Most phenylethanoid glycosides were reported in L. brevituba for the first time. This established method may be employed for comprehensive quality control of L. brevituba. \u00a9 2014 Copyright Taylor & Francis Group, LLC.", "author" : [ { "dropping-particle" : "", "family" : "Li", "given" : "Chunting", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Liu", "given" : "Yongqiang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Abdulla", "given" : "Rahima", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Aisa", "given" : "Haji Akber", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Suo", "given" : "Yourui", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Analytical Letters", "id" : "ITEM-1", "issue" : "11", "issued" : { "date-parts" : [ [ "2014" ] ] }, "page" : "1862-1873", "title" : "Determination of Phenylethanoid Glycosides in Lagotis brevituba Maxim. by High-Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry", "type" : "article-journal", "volume" : "47" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Li et al., 2014)", "manualFormatting" : "Li et al. (2014)", "plainTextFormattedCitation" : "(Li et al., 2014)", "previouslyFormattedCitation" : "(Li et al., 2014)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }Li et al. (2014) and ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1007/s11694-016-9389-x", "ISSN" : "21934134", "abstract" : "The purpose of this study was to evaluate and compare the effects of early (EHVOO) and late harvest (LHVOO) dates on phenolic composition of virgin olive oils (VOO) from the Ayvalik (Edremit) variety. Liquid chromatography combined with a diode array detector and ion spray mass spectrometry in the tandem mode (LC-DAD-ESI-MS/MS) with negative ion detections was used for identification of phenolic compounds in the VOOs. A total of 14 phenolic compounds were identified and quan-tified in VOOs, including phenolic alcohols (2), phenolic acids (4), secoiridoids (5), and flavonoids (3). Secoiridoids followed by phenolic alcohols and flavonoids were the most abundant compounds of the VOOs. Among secoiri-doids 3.4-DHPEA-EDA and 3.4-DHPEA-EA were the most dominant compounds. The antioxidant capacity of VOOs was determined by two different methods, including DPPH and ABTS. The antioxidant capacity of LHVOO was found to be higher than that of EHVOO. Additionally, significant linear correlation was confirmed between the phenolic content and antioxidant capacities of VOOs.", "author" : [ { "dropping-particle" : "", "family" : "Kelebek", "given" : "Hasim", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Selli", "given" : "Serkan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kola", "given" : "Osman", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Food Measurement and Characterization", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2017" ] ] }, "page" : "226-235", "publisher" : "Springer US", "title" : "Quantitative determination of phenolic compounds using LC-DAD-ESI-MS/MS in\u00a0cv. Ayvalik olive oils as affected by harvest time", "type" : "article-journal", "volume" : "11" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Kelebek et al., 2017)", "manualFormatting" : "Kelebek et al. (2017)", "plainTextFormattedCitation" : "(Kelebek et al., 2017)", "previouslyFormattedCitation" : "(Kelebek et al., 2017)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }Kelebek et al. (2017), some aspects are discussed below. The phenolic compounds identified in the olive leaf extracts belong to different classes such as iridoid precursors, secoiridoids and derivatives, flavonoids and phenolic acids. Irinoids are compounds from a broad group of monoterpenes as well as glucoside derivatives. The secoiridoid compounds are derived from iridoids by the opening of the pentacyclic ring.Figure 1: Chromatogram obtained by HPLC–DAD of the phenolic compounds from olive leaves extracted at 60°C, 10 MPa, 0.5 mL min-1. Peak 2 was proposed as being ferulic acid. The mass spectrum showed the deprotonated molecule [M–H]??at?m/z 193, and the MS2?spectrum showed a base peak at?m/z?134. The fragment ion at m/z 134 was a result of the loss of the ethoxyl and hydroxyl groups ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/jf302896m", "ISBN" : "0021-8561", "ISSN" : "00218561", "PMID" : "23106267", "abstract" : "The reuse of agronomical residues is a pending goal for sustainable agriculture. Particular residues in olive-oil-producing countries are leaves, wastewater, and olive pomace. Olive leaves and wastewaters have been previously characterized by isolation of the phenolic fraction. However, olive pomace has not yet been qualitatively characterized as a source of phenols. Olive pomace extracts were obtained using superheated solvent extraction using 50:50 (v/v) water/ethanol as a leaching mixture at 160 \u00b0C. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry using a quadrupole-quadrupole-time-of-flight (QqTOF) hybrid mass analyzer (R = 25 000-45 000). Qualitative analysis was supported upon measurement of accurate masses for precursor and product ions as well as their isotopic distribution. Identification was focused on the main families of phenolic compounds present in extra virgin olive oil. The potential of this residue as a rich source of phenols with antioxidant properties has been proven. \u00a9 2012 American Chemical Society.", "author" : [ { "dropping-particle" : "", "family" : "Peralbo-Molina", "given" : "\u00c1ngela", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Priego-Capote", "given" : "Feliciano", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Luque De Castro", "given" : "Mar\u00eda Dolores", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Agricultural and Food Chemistry", "id" : "ITEM-1", "issue" : "46", "issued" : { "date-parts" : [ [ "2012" ] ] }, "page" : "11542-11550", "title" : "Tentative identification of phenolic compounds in olive pomace extracts using liquid chromatography-tandem mass spectrometry with a quadrupole- quadrupole-time-of-flight mass detector", "type" : "article-journal", "volume" : "60" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Peralbo-Molina et al., 2012)", "plainTextFormattedCitation" : "(Peralbo-Molina et al., 2012)", "previouslyFormattedCitation" : "(Peralbo-Molina et al., 2012)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Peralbo-Molina et al., 2012). Peak 3 was tentatively identified as an oleoside-derivative, based on the [M-H]-?at?m/z 571, and the MS2?mass fragment of?m/z?389, suggesting the presence of an oleoside residue in the molecule. Peak 4a was tentatively assigned as oleoside or secologanoside, based on the [M-H]-?at?m/z?389, since both compounds present the same fragmentation pattern and it is not possible to distinguish between them only by MSn analyses ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/jf302896m", "ISBN" : "0021-8561", "ISSN" : "00218561", "PMID" : "23106267", "abstract" : "The reuse of agronomical residues is a pending goal for sustainable agriculture. Particular residues in olive-oil-producing countries are leaves, wastewater, and olive pomace. Olive leaves and wastewaters have been previously characterized by isolation of the phenolic fraction. However, olive pomace has not yet been qualitatively characterized as a source of phenols. Olive pomace extracts were obtained using superheated solvent extraction using 50:50 (v/v) water/ethanol as a leaching mixture at 160 \u00b0C. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry using a quadrupole-quadrupole-time-of-flight (QqTOF) hybrid mass analyzer (R = 25 000-45 000). Qualitative analysis was supported upon measurement of accurate masses for precursor and product ions as well as their isotopic distribution. Identification was focused on the main families of phenolic compounds present in extra virgin olive oil. The potential of this residue as a rich source of phenols with antioxidant properties has been proven. \u00a9 2012 American Chemical Society.", "author" : [ { "dropping-particle" : "", "family" : "Peralbo-Molina", "given" : "\u00c1ngela", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Priego-Capote", "given" : "Feliciano", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Luque De Castro", "given" : "Mar\u00eda Dolores", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Agricultural and Food Chemistry", "id" : "ITEM-1", "issue" : "46", "issued" : { "date-parts" : [ [ "2012" ] ] }, "page" : "11542-11550", "title" : "Tentative identification of phenolic compounds in olive pomace extracts using liquid chromatography-tandem mass spectrometry with a quadrupole- quadrupole-time-of-flight mass detector", "type" : "article-journal", "volume" : "60" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1002/pca.2401", "ISBN" : "1099-1565", "ISSN" : "09580344", "PMID" : "22987739", "abstract" : "INTRODUCTION: Olea europaea L. leaves may be considered a cheap, easily available natural source of phenolic compounds. In a previous study we evaluated the possibility of obtaining bioactive phenolic compounds from olive leaves by pressurised liquid extraction (PLE) for their use as natural anti-oxidants. The alimentary use of these kinds of extract makes comprehensive knowledge of their composition essential.\\n\\nOBJECTIVE: To undertake a comprehensive characterisation of two olive-leaf extracts obtained by PLE using high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS).\\n\\nMETHOD: Olive leaves were extracted by PLE using ethanol and water as extraction solvents at 150\u00b0C and 200\u00b0C respectively. Separation was carried out in a HPLC system equipped with a C\u2081\u2088-column working in a gradient elution programme coupled to ESI-QTOF-MS operating in negative ion mode.\\n\\nRESULTS: This analytical platform was able to detect 48 compounds and tentatively identify 31 different phenolic compounds in these extracts, including secoiridoids, simple phenols, flavonoids, cinnamic-acid derivatives and benzoic acids. Lucidumoside C was also identified for the first time in olive leaves.\\n\\nCONCLUSION: The coupling of HPLC-ESI-QTOF-MS led to the in-depth characterisation of the olive-leaf extracts on the basis of mass accuracy, true isotopic pattern and tandem mass spectrometry (MS/MS) spectra. We may conclude therefore that this analytical tool is very valuable in the study of phenolic compounds in plant matrices.", "author" : [ { "dropping-particle" : "", "family" : "Quirantes-Pin\u00e9", "given" : "Rosa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lozano-S\u00e1nchez", "given" : "Jes\u00fas", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Herrero", "given" : "Miguel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ib\u00e1\u00f1ez", "given" : "Elena", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Segura-Carretero", "given" : "Antonio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fern\u00e1ndez-Guti\u00e9rrez", "given" : "Alberto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Phytochemical Analysis", "id" : "ITEM-2", "issue" : "3", "issued" : { "date-parts" : [ [ "2013" ] ] }, "page" : "213-223", "title" : "HPLC-ESI-QTOF-MS as a powerful analytical tool for characterising phenolic compounds in olive-leaf extracts", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Peralbo-Molina et al., 2012; Quirantes-Pin\u00e9 et al., 2013)", "plainTextFormattedCitation" : "(Peralbo-Molina et al., 2012; Quirantes-Pin\u00e9 et al., 2013)", "previouslyFormattedCitation" : "(Peralbo-Molina et al., 2012; Quirantes-Pin\u00e9 et al., 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Peralbo-Molina et al., 2012; Quirantes-Piné et al., 2013). Peak 5 was tentatively identified as oleoside methyl ester ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/jf302896m", "ISBN" : "0021-8561", "ISSN" : "00218561", "PMID" : "23106267", "abstract" : "The reuse of agronomical residues is a pending goal for sustainable agriculture. Particular residues in olive-oil-producing countries are leaves, wastewater, and olive pomace. Olive leaves and wastewaters have been previously characterized by isolation of the phenolic fraction. However, olive pomace has not yet been qualitatively characterized as a source of phenols. Olive pomace extracts were obtained using superheated solvent extraction using 50:50 (v/v) water/ethanol as a leaching mixture at 160 \u00b0C. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry using a quadrupole-quadrupole-time-of-flight (QqTOF) hybrid mass analyzer (R = 25 000-45 000). Qualitative analysis was supported upon measurement of accurate masses for precursor and product ions as well as their isotopic distribution. Identification was focused on the main families of phenolic compounds present in extra virgin olive oil. The potential of this residue as a rich source of phenols with antioxidant properties has been proven. \u00a9 2012 American Chemical Society.", "author" : [ { "dropping-particle" : "", "family" : "Peralbo-Molina", "given" : "\u00c1ngela", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Priego-Capote", "given" : "Feliciano", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Luque De Castro", "given" : "Mar\u00eda Dolores", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Agricultural and Food Chemistry", "id" : "ITEM-1", "issue" : "46", "issued" : { "date-parts" : [ [ "2012" ] ] }, "page" : "11542-11550", "title" : "Tentative identification of phenolic compounds in olive pomace extracts using liquid chromatography-tandem mass spectrometry with a quadrupole- quadrupole-time-of-flight mass detector", "type" : "article-journal", "volume" : "60" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1002/pca.2401", "ISBN" : "1099-1565", "ISSN" : "09580344", "PMID" : "22987739", "abstract" : "INTRODUCTION: Olea europaea L. leaves may be considered a cheap, easily available natural source of phenolic compounds. In a previous study we evaluated the possibility of obtaining bioactive phenolic compounds from olive leaves by pressurised liquid extraction (PLE) for their use as natural anti-oxidants. The alimentary use of these kinds of extract makes comprehensive knowledge of their composition essential.\\n\\nOBJECTIVE: To undertake a comprehensive characterisation of two olive-leaf extracts obtained by PLE using high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS).\\n\\nMETHOD: Olive leaves were extracted by PLE using ethanol and water as extraction solvents at 150\u00b0C and 200\u00b0C respectively. Separation was carried out in a HPLC system equipped with a C\u2081\u2088-column working in a gradient elution programme coupled to ESI-QTOF-MS operating in negative ion mode.\\n\\nRESULTS: This analytical platform was able to detect 48 compounds and tentatively identify 31 different phenolic compounds in these extracts, including secoiridoids, simple phenols, flavonoids, cinnamic-acid derivatives and benzoic acids. Lucidumoside C was also identified for the first time in olive leaves.\\n\\nCONCLUSION: The coupling of HPLC-ESI-QTOF-MS led to the in-depth characterisation of the olive-leaf extracts on the basis of mass accuracy, true isotopic pattern and tandem mass spectrometry (MS/MS) spectra. We may conclude therefore that this analytical tool is very valuable in the study of phenolic compounds in plant matrices.", "author" : [ { "dropping-particle" : "", "family" : "Quirantes-Pin\u00e9", "given" : "Rosa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lozano-S\u00e1nchez", "given" : "Jes\u00fas", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Herrero", "given" : "Miguel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ib\u00e1\u00f1ez", "given" : "Elena", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Segura-Carretero", "given" : "Antonio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fern\u00e1ndez-Guti\u00e9rrez", "given" : "Alberto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Phytochemical Analysis", "id" : "ITEM-2", "issue" : "3", "issued" : { "date-parts" : [ [ "2013" ] ] }, "page" : "213-223", "title" : "HPLC-ESI-QTOF-MS as a powerful analytical tool for characterising phenolic compounds in olive-leaf extracts", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Peralbo-Molina et al., 2012; Quirantes-Pin\u00e9 et al., 2013)", "plainTextFormattedCitation" : "(Peralbo-Molina et al., 2012; Quirantes-Pin\u00e9 et al., 2013)", "previouslyFormattedCitation" : "(Peralbo-Molina et al., 2012; Quirantes-Pin\u00e9 et al., 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Peralbo-Molina et al., 2012; Quirantes-Piné et al., 2013). Peak 4b was tentatively identified as verbascoside due to the [M-H]-?at?m/z?623 and consecutive losses of 162 u (m/z 461) and 146 u (m/z 315) observed in the MS2 and MS3 spectra, respectively. Peralbo-Molina et al. (2012) firstly attributed the fragment at m/z 461 to the loss of one glucose moiety ([M-H-162]-); however, Li et al. (2014) found out by LC-QqTOF analysis that this product ion was a result of neutral loss of a caffeoyl rather than a hexose moiety. The product ion at m/z 315 ([M-H-162-146]-) in the MS3 spectrum corresponded to the loss of one rhamnose moiety from the m/z 461 MS2 fragment ion. Peak 7 was identified as oleuropein. It presented the same retention time, MS spectrum, and MS2?and MS3 fragmentation patterns as authentic oleuropein standard. Peak 9 was tentatively identified as an oleuropein isomer since it presented the same spectroscopic characteristics than oleuropein (peak 7). Peak 10 was assigned as being ligstroside. The mass spectrum showed the [M – H]??at?m/z?523 and the MS2?spectrum showed a base peak at?m/z?361 [M–H–162]?, corresponding to the loss of one glucose moiety ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1002/pca.2401", "ISBN" : "1099-1565", "ISSN" : "09580344", "PMID" : "22987739", "abstract" : "INTRODUCTION: Olea europaea L. leaves may be considered a cheap, easily available natural source of phenolic compounds. In a previous study we evaluated the possibility of obtaining bioactive phenolic compounds from olive leaves by pressurised liquid extraction (PLE) for their use as natural anti-oxidants. The alimentary use of these kinds of extract makes comprehensive knowledge of their composition essential.\\n\\nOBJECTIVE: To undertake a comprehensive characterisation of two olive-leaf extracts obtained by PLE using high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS).\\n\\nMETHOD: Olive leaves were extracted by PLE using ethanol and water as extraction solvents at 150\u00b0C and 200\u00b0C respectively. Separation was carried out in a HPLC system equipped with a C\u2081\u2088-column working in a gradient elution programme coupled to ESI-QTOF-MS operating in negative ion mode.\\n\\nRESULTS: This analytical platform was able to detect 48 compounds and tentatively identify 31 different phenolic compounds in these extracts, including secoiridoids, simple phenols, flavonoids, cinnamic-acid derivatives and benzoic acids. Lucidumoside C was also identified for the first time in olive leaves.\\n\\nCONCLUSION: The coupling of HPLC-ESI-QTOF-MS led to the in-depth characterisation of the olive-leaf extracts on the basis of mass accuracy, true isotopic pattern and tandem mass spectrometry (MS/MS) spectra. We may conclude therefore that this analytical tool is very valuable in the study of phenolic compounds in plant matrices.", "author" : [ { "dropping-particle" : "", "family" : "Quirantes-Pin\u00e9", "given" : "Rosa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lozano-S\u00e1nchez", "given" : "Jes\u00fas", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Herrero", "given" : "Miguel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ib\u00e1\u00f1ez", "given" : "Elena", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Segura-Carretero", "given" : "Antonio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fern\u00e1ndez-Guti\u00e9rrez", "given" : "Alberto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Phytochemical Analysis", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "2013" ] ] }, "page" : "213-223", "title" : "HPLC-ESI-QTOF-MS as a powerful analytical tool for characterising phenolic compounds in olive-leaf extracts", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Quirantes-Pin\u00e9 et al., 2013)", "plainTextFormattedCitation" : "(Quirantes-Pin\u00e9 et al., 2013)", "previouslyFormattedCitation" : "(Quirantes-Pin\u00e9 et al., 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Quirantes-Piné et al., 2013). Among the flavonoids tentatively identified in the olive extracts, there is luteolin (peak 18), luteolin-7-O-rutinoside (peak 6a), luteolin hexoside isomers (peaks 6b, 8 and 11), rutin (peak 6c) and quercetin (peak 15). Identification of these compounds was based on their mass spectra and fragmentation patterns, mainly neutral loss of sugar moieties or typical aglycone fragmentation, compared to the literature data ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1021/jf302896m", "ISBN" : "0021-8561", "ISSN" : "00218561", "PMID" : "23106267", "abstract" : "The reuse of agronomical residues is a pending goal for sustainable agriculture. Particular residues in olive-oil-producing countries are leaves, wastewater, and olive pomace. Olive leaves and wastewaters have been previously characterized by isolation of the phenolic fraction. However, olive pomace has not yet been qualitatively characterized as a source of phenols. Olive pomace extracts were obtained using superheated solvent extraction using 50:50 (v/v) water/ethanol as a leaching mixture at 160 \u00b0C. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry using a quadrupole-quadrupole-time-of-flight (QqTOF) hybrid mass analyzer (R = 25 000-45 000). Qualitative analysis was supported upon measurement of accurate masses for precursor and product ions as well as their isotopic distribution. Identification was focused on the main families of phenolic compounds present in extra virgin olive oil. The potential of this residue as a rich source of phenols with antioxidant properties has been proven. \u00a9 2012 American Chemical Society.", "author" : [ { "dropping-particle" : "", "family" : "Peralbo-Molina", "given" : "\u00c1ngela", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Priego-Capote", "given" : "Feliciano", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Luque De Castro", "given" : "Mar\u00eda Dolores", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Agricultural and Food Chemistry", "id" : "ITEM-1", "issue" : "46", "issued" : { "date-parts" : [ [ "2012" ] ] }, "page" : "11542-11550", "title" : "Tentative identification of phenolic compounds in olive pomace extracts using liquid chromatography-tandem mass spectrometry with a quadrupole- quadrupole-time-of-flight mass detector", "type" : "article-journal", "volume" : "60" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1007/s11694-016-9389-x", "ISSN" : "21934134", "abstract" : "The purpose of this study was to evaluate and compare the effects of early (EHVOO) and late harvest (LHVOO) dates on phenolic composition of virgin olive oils (VOO) from the Ayvalik (Edremit) variety. Liquid chromatography combined with a diode array detector and ion spray mass spectrometry in the tandem mode (LC-DAD-ESI-MS/MS) with negative ion detections was used for identification of phenolic compounds in the VOOs. A total of 14 phenolic compounds were identified and quan-tified in VOOs, including phenolic alcohols (2), phenolic acids (4), secoiridoids (5), and flavonoids (3). Secoiridoids followed by phenolic alcohols and flavonoids were the most abundant compounds of the VOOs. Among secoiri-doids 3.4-DHPEA-EDA and 3.4-DHPEA-EA were the most dominant compounds. The antioxidant capacity of VOOs was determined by two different methods, including DPPH and ABTS. The antioxidant capacity of LHVOO was found to be higher than that of EHVOO. Additionally, significant linear correlation was confirmed between the phenolic content and antioxidant capacities of VOOs.", "author" : [ { "dropping-particle" : "", "family" : "Kelebek", "given" : "Hasim", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Selli", "given" : "Serkan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kola", "given" : "Osman", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of Food Measurement and Characterization", "id" : "ITEM-2", "issue" : "1", "issued" : { "date-parts" : [ [ "2017" ] ] }, "page" : "226-235", "publisher" : "Springer US", "title" : "Quantitative determination of phenolic compounds using LC-DAD-ESI-MS/MS in\u00a0cv. Ayvalik olive oils as affected by harvest time", "type" : "article-journal", "volume" : "11" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1002/pca.2401", "ISBN" : "1099-1565", "ISSN" : "09580344", "PMID" : "22987739", "abstract" : "INTRODUCTION: Olea europaea L. leaves may be considered a cheap, easily available natural source of phenolic compounds. In a previous study we evaluated the possibility of obtaining bioactive phenolic compounds from olive leaves by pressurised liquid extraction (PLE) for their use as natural anti-oxidants. The alimentary use of these kinds of extract makes comprehensive knowledge of their composition essential.\\n\\nOBJECTIVE: To undertake a comprehensive characterisation of two olive-leaf extracts obtained by PLE using high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS).\\n\\nMETHOD: Olive leaves were extracted by PLE using ethanol and water as extraction solvents at 150\u00b0C and 200\u00b0C respectively. Separation was carried out in a HPLC system equipped with a C\u2081\u2088-column working in a gradient elution programme coupled to ESI-QTOF-MS operating in negative ion mode.\\n\\nRESULTS: This analytical platform was able to detect 48 compounds and tentatively identify 31 different phenolic compounds in these extracts, including secoiridoids, simple phenols, flavonoids, cinnamic-acid derivatives and benzoic acids. Lucidumoside C was also identified for the first time in olive leaves.\\n\\nCONCLUSION: The coupling of HPLC-ESI-QTOF-MS led to the in-depth characterisation of the olive-leaf extracts on the basis of mass accuracy, true isotopic pattern and tandem mass spectrometry (MS/MS) spectra. We may conclude therefore that this analytical tool is very valuable in the study of phenolic compounds in plant matrices.", "author" : [ { "dropping-particle" : "", "family" : "Quirantes-Pin\u00e9", "given" : "Rosa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lozano-S\u00e1nchez", "given" : "Jes\u00fas", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Herrero", "given" : "Miguel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ib\u00e1\u00f1ez", "given" : "Elena", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Segura-Carretero", "given" : "Antonio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fern\u00e1ndez-Guti\u00e9rrez", "given" : "Alberto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Phytochemical Analysis", "id" : "ITEM-3", "issue" : "3", "issued" : { "date-parts" : [ [ "2013" ] ] }, "page" : "213-223", "title" : "HPLC-ESI-QTOF-MS as a powerful analytical tool for characterising phenolic compounds in olive-leaf extracts", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] } ], "mendeley" : { "formattedCitation" : "(Kelebek et al., 2017; Peralbo-Molina et al., 2012; Quirantes-Pin\u00e9 et al., 2013)", "plainTextFormattedCitation" : "(Kelebek et al., 2017; Peralbo-Molina et al., 2012; Quirantes-Pin\u00e9 et al., 2013)", "previouslyFormattedCitation" : "(Kelebek et al., 2017; Peralbo-Molina et al., 2012; Quirantes-Pin\u00e9 et al., 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }(Kelebek et al., 2017; Peralbo-Molina et al., 2012; Quirantes-Piné et al., 2013).ConclusionThe operational conditions evaluated in this study allow to evaluate the extraction of active compounds from the olive leaf, mainly oleuropein, for milder temperatures, high pressures and continuous flow concerning the information available in the literature regarding PLE extraction.The temperature was the most active operational parameter to obtain an extract of olive leaves by PLE in a continuous flow. In the 60 ?C condition, the highest yields (14.5 to 15.1%) were obtained. The pressure and flow rate did not have significant influence; therefore, lower flow (0.5 mL min-1) and pressure (10 MPa) levels can be used in the extraction to promote energy and solvent savings. The phenolic compounds profile identified in the olive leaf extracts belong to different classes such as iridoid precursors, secoiridoids and derivatives, flavonoids and phenolic acids. The parameters of temperature, pressure and solvent flow did not influence the content of oleuropein under the experimental conditions of this study. The antioxidant activity was higher in extracts obtained at a temperature of 30 ?C, the flow rate of 0.5 mL min-1 and pressure of 10 MPa.The extraction with pressurized ethanol has low toxicity to the environment and is promising to obtain oleuropein and other phenolic compounds from olive leaves. The best condition to obtain an extract with the highest yield and total phenolic compounds and suitable oleuropein and antioxidant capacity would be 60 oC, 10 MPa, and 0.5 mL min-1.AcknowledgmentsThe authors thank S?o Paulo Research Foundation (FAPESP) (Grants No. 2017/03614-0).ReferencesADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY Al-Azzawie, H.F., Alhamdani, M.S.S., 2006. Hypoglycemic and antioxidant effect of oleuropein in alloxan-diabetic rabbits. Life Sci. 78, 1371–1377.Albarelli J., Vardanega R., Wilkins N., Santos D., Marechal F., Meireles M., 2016, Economical analysis of a pressurized fluid-based process applied for phytochemicals recovery in a sustainable biorefinery concept for brazilian ginseng roots, Chemical Engineering Transactions, 50, 355-360Bulotta, S., Celano, M., Lepore, S.M., Montalcini, T., Pujia, A., Russo, D., 2014. 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