Hepatoprotective species from the Chilean medicinal flora: Junellia spathulata (Verbenaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Chilean population relies on medicinal plants for treating a wide range of illnesses, especially those of the gastrointestinal system. Junellia spathulata (Gillies & Hook.) Moldenke var. spathulata (Verbenaceae), called as "verbena-azul-de-cordilleira", is a medicinal plant native to Argentina and Chile traditionally used for treating digestive disorders. Although the species of the genus are important as therapeutic resources for the Andean population, the plants are very scarcely studied. AIMS OF THE STUDY: The purpose of the present study was to find out the main constituents and investigate the protective effect of J. spathulata against oxidative stress induced by the potent oxidant 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) in human hepatoblastoma cells. MATERIALS AND METHODS: The crude methanol extract of J. spathulata and an iridoid obtained by chromatographic processes were tested to access the hepatoprotective effect and cytotoxicity in HepG2 cell. In addition, the reducing power of the samples and their ability to scavenge free radicals were evaluated using FRAP and ORAC assay systems. RESULTS: The iridoid asperuloside, the main compound of the crude methanol extract of J. spathulata, was isolated and identified by means of NMR analysis. The crude methanol extract of J. spathulata and asperuloside protected HepG2 cells against oxidative damage triggered by AAPH-derived free radicals. This effect can be credited to the ability of the extract and asperuloside to protect the liver cells from chemical-induced injury, which might be correlated to their free radical scavenging potential. CONCLUSIONS: This study experimentally evidenced the ethnopharmacological usefulness of J. spathulata as a treatment of digestive disorders. Our result could stimulate further investigations of hepatoprotective agents in other Chilean Junellia species.

Green Route for the Isolation and Purification of Hyrdoxytyrosol, Tyrosol, Oleacein and Oleocanthal from Extra Virgin Olive Oil.

Extra virgin olive oil (EVOO) phenols represent a significant part of the intake of antioxidants and bioactive compounds in the Mediterranean diet. In particular, hydroxytyrosol (HTyr), tyrosol (Tyr), and the secoiridoids oleacein and oleocanthal play central roles as anti-inflammatory, neuro-protective and anti-cancer agents. These compounds cannot be easily obtained via chemical synthesis, and their isolation and purification from EVOO is cumbersome. Indeed, both processes involve the use of large volumes of organic solvents, hazardous reagents and several chromatographic steps. In this work we propose a novel optimized procedure for the green extraction, isolation and purification of HTyr, Tyr, oleacein and oleocanthal directly from EVOO, by using a Natural Deep Eutectic Solvent (NaDES) as an extracting phase, coupled with preparative high-performance liquid chromatography. This purification method allows the total recovery of the four components as single pure compounds directly from EVOO, in a rapid, economic and ecologically sustainable way, which utilizes biocompatible reagents and strongly limits the use or generation of hazardous substances.

(-)-Oleocanthal as a Dual c-MET-COX2 Inhibitor for the Control of Lung Cancer.

Lung cancer (LC) represents the topmost mortality-causing cancer in the U.S. LC patients have overall poor survival rate with limited available treatment options. Dysregulation of the mesenchymal epithelial transition factor (c-MET) and cyclooxygenase 2 (COX2) initiates aggressive LC profile in a subset of patients. The Mediterranean extra-virgin olive oil (EVOO)-rich diet already documented to reduce multiple malignancies incidence. (-)-Oleocanthal (OC) is a naturally occurring phenolic secoiridoid exclusively occurring in EVOO and showed documented anti-breast and other cancer activities via targeting c-MET. This study shows the novel ability of OC to suppress LC progression and metastasis through dual targeting of c-MET and COX-2. Western blot analysis and COX enzymatic assay showed significant reduction in the total and activated c-MET levels and inhibition of COX1/2 activity in the lung adenocarcinoma cells A549 and NCI-H322M, in vitro. In addition, OC treatment caused a dose-dependent inhibition of the HGF-induced LC cells migration. Daily oral treatment with 10 mg/kg OC for 8 weeks significantly suppressed the LC A549-Luc progression and prevented metastasis to brain and other organs in a nude mouse tail vein injection model. Further, microarray data of OC-treated lung tumors showed a distinct gene signature that confirmed the dual targeting of c-MET and COX2. Thus, the EVOO-based OC is an effective lead with translational potential for use as a prospective nutraceutical to control LC progression and metastasis.

Influence of Harvest Time and Malaxation Conditions on the Concentration of Individual Phenols in Extra Virgin Olive Oil Related to Its Healthy Properties.

The phenolic fraction of the extra virgin olive oil (EVOO) has been studied over the past two decades because of its important health protective properties. Numerous studies have been performed in order to clarify the most crucial factors that affect the concentration of the EVOO's phenolic fraction and many contradictory results have been reported. Having as target to maximize the phenolic content of EVOO and its healthy properties we investigated the impact of harvest time, malaxation temperature, and malaxation duration on the concentration of individual phenols in extra virgin olive oil. Olive oil was prepared in a lab-scale olive mill from different varieties in Greece. The extraction process for cultivar (cv) Koroneiki samples was performed at five different harvest periods from the same trees with three different malaxation temperatures and five different malaxation duration times (N = 75). Similar types of experiments were also performed for other varieties: cv Athenolia (N = 20), cv Olympia (N = 3), cv Kalamata (N = 3), and cv Throubolia Aegean (N=3) in order to compare the changes in the phenolic profile during malaxation. The quantitative analysis of the olive oil samples with NMR showed that the total phenolic content has a negative correlation with the ripening degree and the malaxation time. The NMR data we collected helped us to quantitate not only the total phenolic content but also the concentration of the major phenolic compounds such as oleocanthal, oleacein, oleokoronal, and oleomissional. We noticed different trends for the concentration of these phenols during malaxation process and for different malaxation temperatures. The different trends of the concentration of the individual phenols during malaxation and the completely different behavior of each variety revealed possible biosynthetic formation steps for oleocanthal and oleacein and may explain the discrepancies reported from previous studies.

Safety Evaluations of Single Dose of the Olive Secoiridoid S-(-)-Oleocanthal in Swiss Albino Mice.

Epidemiological and clinical studies compellingly showed the ability of Mediterranean diet rich in extra-virgin olive oil (EVOO) to reduce multiple diseases such as cancer, cardiovascular diseases, and aging cognitive functions decline. The S-(-)-Oleocanthal (OC) is a minor phenolic secoiridoid exclusively found in extra-virgin olive oil (EVOO). OC recently gained notable research attention due to its excellent in vitro and in vivo biological effects against multiple cancers, inflammations, and Alzheimer's disease. However, OC safety has not been comprehensively studied yet. This study reports for the first time the detailed safety of oral single OC dose in Swiss albino mice, applying the OECD 420 procedure. Male and female Swiss albino mice (n = 10) were orally treated with a single OC dose of either 10, 250, or 500 mg/kg bodyweight or equivalent volumes of distilled water. Mice fed a regular diet, and carefully observed for 14 days. Further, mice were then sacrificed, blood samples, and organs were collected and subjected to hematological, biochemical, and histological examinations. OC 10 mg/kg oral dose appears to be without adverse effects. Further, 250 mg/kg OC, p.o., is suggested as a possible upper dose for preclinical studies in the future.

A comprehensive study of oleuropein aglycone isomers in olive oil by enzymatic/chemical processes and liquid chromatography-Fourier transform mass spectrometry integrated by H/D exchange.

A comprehensive structural characterization of the complex family of isomeric forms related to Oleuropein aglycone (OA) detected in virgin olive oil (VOO) was performed by reverse phase liquid chromatography with electrospray ionization and Fourier-transform mass spectrometry (RPLC-ESI-FTMS), integrated by enzymatic/chemical reactions performed on Oleuropein, the natural precursor of OA. First, some of the OA-related isomers typically observed in VOO extracts were generated upon enzymatic hydrolysis of the glycosidic linkage of Oleuropein. This step mimicked the process occurring during olive drupes crushing in the first stage of oil production. The incubation of the enzymatic reaction mixture at a more acidic pH was subsequently performed, to simulate the conditions of olive paste malaxation during oil production. As a result, further isomeric forms were generated and the complex chromatographic profile typically observed for OA in olive oil extracts, including at least 13 different peaks/bands/groups of peaks, was carefully reproduced. Each of those chromatographic features could be subsequently assigned to specific types of OA-related isomers, belonging to one of four structurally different classes. Specifically, diastereoisomers/geometrical isomers corresponding to two different types of open-structure forms and to as many types of closed-structure, di-hydropyranic forms of OA, characterized by the presence of one or two carbonyl groups, according to the case, were evidenced. In addition, the presence of stable enolic/dienolic tautomers, providing an indirect structural confirmation for some OA isomers, was ascertained through RPLC-ESI-FTMS analyses performed under H/D exchange conditions, i.e. in the presence of deuterated water as one of the mobile phase solvents.