Oleuropein was purchased from Extrasynthese and deglycosilated by treatment with almond β-glycosidase (EC 3.2.1.21, Fluka, Sigma-Aldrich, St. Louis, MI, USA), as previously described [45 (link)]. Briefly, a 10 mM solution of Oleuropein in 310 μL of 0.1 M sodium phosphate buffer, pH 7.0, was incubated with 8.9 I.U. of β-glycosidase overnight at room temperature. The reaction mixture was centrifuged at 18,000 rpm for 10 min to precipitate OleA, which was resuspended in dimethylsulfoxide (DMSO) in stocks at 50 mM concentration. The complete Oleuropein deglycosylation was confirmed by assaying the glucose released in the supernatant with a Glucose (HK) Assay kit (Sigma-Aldrich, St. Louis, MI, USA). Stocks of OleA were kept frozen protected from light and were used within the same day once opened.
Oleuropein
Manufactured by Merck Group
Sourced in United States, Germany, Italy, France, Spain
Oleuropein is a chemical compound found in the leaves and fruit of olive trees. It is a complex phenolic compound with various biological properties. Oleuropein is used in laboratory research applications, but a detailed description of its core function cannot be provided in a concise, unbiased, and factual manner without risk of extrapolation or interpretation.
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Lab products found in correlation
Hydroxytyrosol, by Merck Group (30 mentions)
Gallic acid, by Merck Group (27 mentions)
Tyrosol, by Merck Group (25 mentions)
P coumaric acid, by Merck Group (23 mentions)
Caffeic acid, by Merck Group (22 mentions)
Apigenin, by Merck Group (22 mentions)
Luteolin, by Merck Group (22 mentions)
Ferulic acid, by Merck Group (21 mentions)
Vanillic acid, by Merck Group (19 mentions)
Methanol, by Merck Group (18 mentions)
Rutin, by Merck Group (16 mentions)
Pinoresinol, by Merck Group (16 mentions)
Acetonitrile, by Merck Group (16 mentions)
Formic acid, by Merck Group (13 mentions)
Quercetin, by Merck Group (12 mentions)
Chlorogenic acid, by Merck Group (11 mentions)
Folin ciocalteu reagent, by Merck Group (11 mentions)
Sodium carbonate, by Merck Group (10 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (10 mentions)
Verbascoside, by Merck Group (10 mentions)
90 protocols using oleuropein
1
Enzymatic Deglycosylation of Oleuropein
2
Oleuropein's Impact on FA-Induced Steatosis
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The human hepatoma cell line, HepG2, (ATCC cat. HB-8065) were purchased from American Type Culture Collection (ATCC, Manassas, VA). HepG2 were cultivated as previously described by Santini and colleagues [29 (link)]. Long-chain FAs, palmitic acid (PA; 16:0) and oleic acid (OA; 18:1) (Sigma-Aldrich, Milan, Italy) were dissolved in methanol (MetOH) 99%. Steatosis was induced as previously described by Ricchi et al. [36 (link)]. Briefly, culture medium was supplemented with a solution of FAs (0.16 mM PA and 0.33 mM OA). Cells incubated with MetOH were considered as control.
After 24 h, Oleuropein was added to HepG2 cells at the following concentrations: 50, 100 and 200 μM. Oleuropein (Sigma-Aldrich cat. 12247, Milan, Italy).
After 24 h, Oleuropein was added to HepG2 cells at the following concentrations: 50, 100 and 200 μM. Oleuropein (Sigma-Aldrich cat. 12247, Milan, Italy).
3
Breast Cancer Cell Line Analyses with Fatty Acids and EVOO Compounds
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For in vitro analyses, we used the MCF-7 cell line, representing the molecular subtype of human breast cancer Luminal A, and MDA-MB-231 cells, representing the triple-negative subtype. Cell lines were obtained from the American Type Culture Collection (ATCC; LGC Standards, Middlesex, UK). MCF-7 cells were grown in EMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with 10% fetal bovine serum (FBS; Gibco) and 0.01 mg/mL of insulin. MDA-MB-231 cells were grown in DMEM (Gibco) and supplemented with 10% FBS. Cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The cell cultures were subjected to Mycoplasma screen tests periodically.
For fatty acid treatments, oleic acid- or linoleic acid-albumin from bovine serum (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) were used at 0 µM (control), 1 µM, 10 µM, 100 µM, or 1 mM concentration. All the solutions contained 0.1 mg/mL of BSA (Sigma-Aldrich).
For treatments with EVOO minor compounds, we used hydroxytyrosol (PHL80152, Sigma Aldrich) at 0 µM (control), 100 µM, 250 µM, and 400 µM; oleuropein (12,247, Sigma Aldrich) at 0 µM (control), 10 µM, 30 µM, and 50 µM; and lutein (L9283, Sigma-Aldrich) at 0 µM (control), 5 µM, 10 µM, and 30 µM [25 (link),26 (link),27 (link)]. All the solutions contained 0.1% of DMSO.
For fatty acid treatments, oleic acid- or linoleic acid-albumin from bovine serum (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) were used at 0 µM (control), 1 µM, 10 µM, 100 µM, or 1 mM concentration. All the solutions contained 0.1 mg/mL of BSA (Sigma-Aldrich).
For treatments with EVOO minor compounds, we used hydroxytyrosol (PHL80152, Sigma Aldrich) at 0 µM (control), 100 µM, 250 µM, and 400 µM; oleuropein (12,247, Sigma Aldrich) at 0 µM (control), 10 µM, 30 µM, and 50 µM; and lutein (L9283, Sigma-Aldrich) at 0 µM (control), 5 µM, 10 µM, and 30 µM [25 (link),26 (link),27 (link)]. All the solutions contained 0.1% of DMSO.
4
Quantification of Phenolic Compounds
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The 2,5-dihydroxybenzoic acid (2,5-DHBA), 3,4-dihydroxybenzoic acid (3,4-DHBA), 4-hydroxybenzoic acid (pHBA), 3-hydroxytyrosol, apigenin, caffeic acid, chrysin, quercetin, naringenin, diosmetin, rutin, luteolin, luteolin-7-O-glucoside, p-coumaric acid, oleuropein and tyrosol were all from Sigma-Aldrich (Merck, St. Louis, MO, USA). Ferulic acid was purchased from Extrasynthese (Genay, France). apigenin-7-O-glucoside and verbascoside were obtained from HWI Analytik GmbH (Rülzheim, Germany). Gallic acid was obtained from Alfa Aesar (Thermo Fisher Scientific, Waltham, MA, USA).
Chemical reagents methanol (LC-MS purity) and acetonitrile (LC-MS purity) were purchased from Honeywell Research Chemicals (Bucharest, Romania). Formic acid (LC-MS purity) was obtained from Sigma-Aldrich, Merck. Milli-Q water was obtained using a Milli-Q water purification system (<0.058 μS/cm, Mili-Q model Pacific TII 12; Barnstead™, Thermo Fisher Scientific).
Chemical reagents methanol (LC-MS purity) and acetonitrile (LC-MS purity) were purchased from Honeywell Research Chemicals (Bucharest, Romania). Formic acid (LC-MS purity) was obtained from Sigma-Aldrich, Merck. Milli-Q water was obtained using a Milli-Q water purification system (<0.058 μS/cm, Mili-Q model Pacific TII 12; Barnstead™, Thermo Fisher Scientific).
5
Analytical Standards for Phenolic Profiling
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Ultrapure water was obtained through a purification system (Millipore Direct-Q UV, Bedford, MA, USA). Formic acid, of LC-MS grade, and ammonium acetate ≥99% were obtained from Fluka (Buchs, Switzerland). Sodium hydroxide >99% and methanol, of LC-MS grade, were acquired from Merck (Darmstadt, Germany). 2-propanol was acquired from Fisher Scientific (Geel, Belgium).
All standards had an analytical grade ≥95%. Epicatechin, ferulic acid, gallic acid, homovanillic acid, oleuropein, pinoresinol, p-coumaric acid, and syringaldehyde were purchased from Sigma-Aldrich (Steinheim, Germany). Apigenin, caffeic acid, tyrosol, and vanillin were acquired from Alfa Aesar (Karlsruhe, Germany). Luteolin and hydroxytyrosol were purchased from Santa Cruz Biotechnologies (Heidelberg, Germany). Syringic acid was obtained from Extrasynthèse (Genay, France). Stock solutions for each analyte (1000 mg/L) were dissolved in methanol and stored at −20 °C. Mixed standard working solutions were prepared every laboratory day diluting the stock solutions with water:methanol (20:80, v/v).
All standards had an analytical grade ≥95%. Epicatechin, ferulic acid, gallic acid, homovanillic acid, oleuropein, pinoresinol, p-coumaric acid, and syringaldehyde were purchased from Sigma-Aldrich (Steinheim, Germany). Apigenin, caffeic acid, tyrosol, and vanillin were acquired from Alfa Aesar (Karlsruhe, Germany). Luteolin and hydroxytyrosol were purchased from Santa Cruz Biotechnologies (Heidelberg, Germany). Syringic acid was obtained from Extrasynthèse (Genay, France). Stock solutions for each analyte (1000 mg/L) were dissolved in methanol and stored at −20 °C. Mixed standard working solutions were prepared every laboratory day diluting the stock solutions with water:methanol (20:80, v/v).
6
Polyphenol Extraction and Quantification
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For extraction, ethanol and sodium hydroxide (NaOH) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and VWR Chemicals (Radnor, PA, USA), respectively. Acetone, methanol and acetonitrile were purchased from PanReac AppliChem (Barcelona, Spain). Folin and Ciocalteu’s phenol reagent, sodium carbonate, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), and standards of oleuropein, luteolin 7-O-glucoside and gallic acid were purchased from Sigma-Aldrich. Ultrapure water was obtained by a Milli-Q system (Millipore, Bedford, MA, USA).
7
Cytotoxicity Evaluation of Natural Compounds
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Acetonitrile for the HPLC-DAD-MS analysis was purchased by Merck (Darmstadt, Germany), while water was purified with a Direct-Q UV system by Millipore (Darmstadt, Germany). Chlorogenic acid, rutin and oleuropein (analytical purity) were purchased from Sigma Aldrich (Darmstadt, Germany). All other chemicals used were purchased from Alfa-Aesar, Karlsruhe, Germany. The cytotoxicity was tested on two tumoral cell lines: murine melanoma cells (B16F10 cells) and human cancer cell line (HeLa). The selected cell lines were obtained from American Type Cell Collection (ATCC) (Manassas, VA, USA). The B16F10 cells were maintained in RPMI 1640 medium (Sigma Aldrich, Darmstadt, Germany) supplemented with 10% fetal bovine serum (EuroClone, MI, Pero, Italy) and 1% Antibiotic-Antimycotic 100× (Sigma Aldrich, Darmstadt, Germany). Hela cells were maintained in DMEM/F-12 medium (Gibco Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (EuroClone, MI, Pero, Italy) and 1% Antibiotic-Antimycotic 100× (Sigma Aldrich, Darmstadt, Germany). The two cell lines were cultured in a 5% CO2 incubator (Advantage-Lab, Schilde, Belgium) at 37 °C in a humidified atmosphere. Cisplatin (Ebewe Pharma Ges.m.b. H. Nfg. KG, Unterach am Attersee, Austria) was included as standard positive control for cytotoxicity assay.
8
Antioxidant Compound Analysis Protocol
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Ethanol and mEthanol were provided from Merck (Darmstadt, Germany). Eighteen mΩ deionised water (from a Human Power I water purification system) was used for analysis. Oleuropein, 2,9-dimethyl-1,10-phenanthroline (neocuproine), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®), Folin–Ciocalteu reagent, sodium carbonate, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), were purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Extraction and Analysis of Phenolic Compounds
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Amberlite® XAD7HP (DuPont, Wilmington, DE, USA), chromatographic grade chemicals of hexane, water, acetic acid, sodium acetate trihydrate, methanol, ethanol, acetonitrile and analytical grade of chemicals of 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH), Trolox®, 2,4,6-tripirydyl-Striazine (TPTZ) and ferric chloride were purchased from Fisher Scientific (Waltham, MA, USA). In addition, 96–98% (g/g) concentrated sulfuric acid, Folin–Ciocalteu reagents, sodium carbonate, phenolic compound standards of gallic acid, hydroxytyrosol, tyrosol, 4-hydroxyphenylacetic acid (4-HPA), vanillic acid, vanillin, o-coumaric acid, oleuropein, pinoresinol, cinnamic acid, caffeic acid, p-coumaric acid, ferulic acid, apigenin-7-glucoside, apigenin, luteolin-7-glucoside and luteolin were purchased from Sigma-Aldrich (St. Louis, MI, USA). Verbascoside was bought from the HWI group (Ruelzhelm, Germany). Rutin was bought from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany).
10
Profiling Compounds in CareVid™ Tea Powder
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The nine compounds (1 –9 ) were determined using HPLC-MS to be present in CareVidTM as described previously [1 (link)]. Extracts were prepared initially from 20 g each of CareVidTM Tea powder. The extracts were methylene chloride, ethanol, methanol, water, acidified water (0.1 M HCl), and steeping in hot water (>90 °C). Metabolic profiling of the extracts was performed by UHPLC-UV-HRMS/MS on a Vanquish UHPLC system (Thermo Scientific, Waltham, MA, USA) coupled to a 100 Hz photodiode array detector (PDA) and an Orbitrap Fusion Tribrid (Thermo Scientific) high-resolution tandem mass spectrometer (Thermo Scientific, Waltham, MA, USA). A detailed protocol that was followed is described in Rotich et al., 2021 (Figure 1 ). For the assay, pure standards of pellitorine (1 ), oleuropein (2 ) and ellagic acid (9 ), urolithin A (10 ), and urolithin B (11 ) were bought from Sigma Aldrich, whereas pure standards of magnoflorine (3 ) earlier isolated from the seed cake of Croton megalocarpus, crotocorylifuran (6 ) earlier isolated from Croton megalocarpoides, crotepoxide (4 ) earlier isolated from Croton alienus, ent-kaurane-16β,17-diol (5 ) earlier isolated from Croton haumanianus, and lupeol (7 ) and betulin (8 ) isolated in substantial quantities from CareVidTM were obtained in the Jodrell Laboratory, Kew, UK.
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