Quantitation of Lupinus spp. Quinolizidine Alkaloids by qNMR and Accelerated Debittering with a Resin-Based Protocol.

Quinolizidine alkaloids (QAs) are toxic secondary metabolites of the Lupinus species, the presence of which limits the expansion of lupin beans consumption, despite their high protein content. Evaluation of the level of alkaloids in edible Lupinus species is crucial from a food safety point of view. However, quantitation of QAs is complicated by the fact that not all important alkaloids used for quantitation are commercially available. In this context, we developed a method for the simultaneous quantitation of eight major lupin alkaloids using quantitative NMR spectroscopy (qNMR). Quantitation and analysis were performed in 15 different seed extracts of 11 Lupinus spp. some of which belonged to the same species, with different geographical origins and time of harvest, as well as in all aerial parts of L. pilosus. The mature seeds of L. pilosus were found to be a uniquely rich source of multiflorine. Additionally, we developed a protocol using adsorption or ionic resins for easy, fast, and efficient debittering of the lupine seeds. The protocol was applied to L. albus, leading to a decrease of the time required for alkaloids removal as well as water consumption and to a method for QA isolation from the debittering wastewater.

Acute Antiplatelet Effects of an Oleocanthal-Rich Olive Oil in Type II Diabetic Patients: A Postprandial Study.

Postprandial dysmetabolism is a common entity of type 2 diabetes mellitus (T2DM) and may act as a daily stressor of the already dysfunctional diabetic platelets. This study aims to investigate whether oleocanthal-rich olive oils (OO), incorporated into a carbohydrate-rich meal, can affect postprandial dysmetabolism and platelet aggregation. Oleocanthal is a cyclooxygenase inhibitor with putative antiplatelet properties. In this randomized, single-blinded, crossover study, ten T2DM patients consumed five isocaloric meals containing 120 g white bread combined with: (i) 39 g butter, (ii) 39 g butter and 400 mg ibuprofen, (iii) 40 mL OO (phenolic content < 10 mg/Kg), (iv) 40 mL OO with 250 mg/Kg oleocanthal and (v) 40 mL OO with 500 mg/Kg oleocanthal. Metabolic markers along with ex vivo ADP- and thrombin receptor-activating peptide (TRAP)-induced platelet aggregation were measured before and for 4 h after the meals. The glycemic and lipidemic response was similar between meals. However, a sustained (90-240 min) dose-dependent reduction in platelets' sensitivity to both ADP (50-100%) and TRAP (20-50%) was observed after the oleocanthal meals in comparison to OO or butter meals. The antiplatelet effect of the OO containing 500 mg/Kg oleocanthal was comparable to that of the ibuprofen meal. In conclusion, the consumption of meals containing oleocanthal-rich OO can reduce platelet activity during the postprandial period, irrespective of postprandial hyperglycemia and lipidemia.

A Simple Chiral 1H NMR Method for the Discrimination of (R)- and (S)-Cannabichromene in Complex Natural Mixtures and Their Effects on TRPA1 Activity.

In recent years, the enantiomeric ratio of cannabichromene (CBC) within the cannabis plant has attracted significant attention. Cannabichromene is one of the well-known cannabinoids found in cannabis, along with THC (tetrahydrocannabinol) and CBD (cannabidiol). Cannabichromene exists as a scalemic mixture, meaning it has two enantiomers, (S)-cannabichromene and (R)-cannabichromene, with the ratio between these enantiomers varying among different cannabis strains and even within individual plants. This study presents an accurate and robust chiral NMR method for analyzing cannabichromene's enantiomeric ratio, a well-investigated cannabinoid with numerous pharmacological targets. The use of Pirkle's alcohol as the chiral solvating agent (CSA) or, alternatively, the use of (S)-ibuprofen as a chiral derivatizing agent (CDA) facilitated this analysis. Moreover, the chiral NMR method proves to be a user-friendly tool, easily applicable within any NMR facility, and an expanded investigation of cannabichromene chirality may provide insights into the origin, cultivation, treatment, and processing of Cannabis sativa plants. This study also undertakes a pharmacological examination of the (R)- and (S)-cannabichromenes concerning their most extensively studied pharmacological target, the TRPA1 channels, with the two enantiomers showing the same strong agonistic effect as the racemic mixture.

A Systematic Ex-Vivo Study of the Anti-Proliferative/Cytotoxic Bioactivity of Major Olive Secoiridoids' Double Combinations and of Total Olive Oil Phenolic Extracts on Multiple Cell-Culture Based Cancer Models Highlights Synergistic Effects.

Several individual olive oil phenols (OOPs) and their secoiridoid derivatives have been shown to exert anti-proliferative and pro-apoptotic activity in treatments of human cancer cell lines originating from several tissues. This study evaluated the synergistic anti-proliferative/cytotoxic effects of five olive secoiridoid derivatives (oleocanthal, oleacein, oleuropein aglycone, ligstroside aglycone and oleomissional) in all possible double combinations and of total phenolic extracts (TPEs) on eleven human cancer cell lines representing eight cell-culture-based cancer models. Individual OOPs were used to treat cells for 72 h in half of their EC50 values for each cell line and their synergistic, additive or antagonistic interactions were evaluated by calculating the coefficient for drug interactions (CDI) for each double combination of OOPs. Olive oil TPEs of determined OOPs' content, originating from three different harvests of autochthonous olive cultivars in Greece, were evaluated as an attempt to investigate the efficacy of OOPs to reduce cancer cell numbers as part of olive oil consumption. Most combinations of OOPs showed strong synergistic effect (CDIs < 0.9) in their efficacy, whereas TPEs strongly impaired cancer cell viability, better than most individual OOPs tested herein, including the most resistant cancer cell lines evaluated.

Treatment with the Olive Secoiridoid Oleacein Protects against the Intestinal Alterations Associated with EAE.

Multiple sclerosis (MS) is a CNS inflammatory demyelinating disease. Recent investigations highlight the gut-brain axis as a communication network with crucial implications in neurological diseases. Thus, disrupted intestinal integrity allows the translocation of luminal molecules into systemic circulation, promoting systemic/brain immune-inflammatory responses. In both, MS and its preclinical model, the experimental autoimmune encephalomyelitis (EAE) gastrointestinal symptoms including "leaky gut" have been reported. Oleacein (OLE), a phenolic compound from extra virgin olive oil or olive leaves, harbors a wide range of therapeutic properties. Previously, we showed OLE effectiveness preventing motor defects and inflammatory damage of CNS tissues on EAE mice. The current studies examine its potential protective effects on intestinal barrier dysfunction using MOG35-55-induced EAE in C57BL/6 mice. OLE decreased EAE-induced inflammation and oxidative stress in the intestine, preventing tissue injury and permeability alterations. OLE protected from EAE-induced superoxide anion and accumulation of protein and lipid oxidation products in colon, also enhancing its antioxidant capacity. These effects were accompanied by reduced colonic IL-1ß and TNFα levels in OLE-treated EAE mice, whereas the immunoregulatory cytokines IL-25 and IL-33 remained unchanged. Moreover, OLE protected the mucin-containing goblet cells in colon and the serum levels of iFABP and sCD14, markers that reflect loss of intestinal epithelial barrier integrity and low-grade systemic inflammation, were significantly reduced. These effects on intestinal permeability did not draw significant differences on the abundance and diversity of gut microbiota. However, OLE induced an EAE-independent raise in the abundance of Akkermansiaceae family. Consistently, using Caco-2 cells as an in vitro model, we confirmed that OLE protected against intestinal barrier dysfunction induced by harmful mediators present in both EAE and MS. This study proves that the protective effect of OLE in EAE also involves normalizing the gut alterations associated to the disease.

Direct Quantitation of Phytocannabinoids by One-Dimensional 1H qNMR and Two-Dimensional 1H-1H COSY qNMR in Complex Natural Mixtures.

The widespread use of phytocannabinoids or cannabis extracts as ingredients in numerous types of products, in combination with the legal restrictions on THC content, has created a need for the development of new, rapid, and universal analytical methods for their quantitation that ideally could be applied without separation and standards. Based on previously described qNMR studies, we developed an expanded 1H qNMR method and a novel 2D-COSY qNMR method for the rapid quantitation of ten major phytocannabinoids in cannabis plant extracts and cannabis-based products. The 1H qNMR method was successfully developed for the quantitation of cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabielsoin (CBE), and cannabidivarin (CBDV). Moreover, cannabidivarinic acid (CBDVA) and Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA) can be distinguished from CBDA and Δ9-THCA respectively, while cannabigerovarin (CBGV) and Δ8-tetrahydrocannabivarin (Δ8-THCV) present the same 1H-spectra as CBG and Δ8-THC, respectively. The COSY qNMR method was applied for the quantitation of CBD, CBDA, CBN, CBG/CBGA, and THC/THCA. The two methods were applied for the analysis of hemp plants; cannabis extracts; edible cannabis medium-chain triglycerides (MCT); and hemp seed oils and cosmetic products with cannabinoids. The 1H-NMR method does not require the use of reference compounds, and it requires only a short time for analysis. However, complex extracts in 1H-NMR may have a lot of signals, and quantitation with this method is often hampered by peak overlap, with 2D NMR providing a solution to this obstacle. The most important advantage of the COSY NMR quantitation method was the determination of the legality of cannabis plants, extracts, and edible oils based on their THC/THCA content, particularly in the cases of some samples for which the determination of THC/THCA content by 1H qNMR was not feasible.

New Affordable Methods for Large-Scale Isolation of Major Olive Secoiridoids and Systematic Comparative Study of Their Antiproliferative/Cytotoxic Effect on Multiple Cancer Cell Lines of Different Cancer Origins.

Olive oil phenols (OOPs) are associated with the prevention of many human cancers. Some of these have been shown to inhibit cell proliferation and induce apoptosis. However, no systematic comparative study exists for all the investigated compounds under the same conditions, due to difficulties in their isolation or synthesis. Herein are presented innovative methods for large-scale selective extraction of six major secoiridoids from olive oil or leaves enabling their detailed investigation. The cytotoxic/antiproliferative bioactivity of these six compounds was evaluated on sixteen human cancer cell lines originating from eight different tissues. Cell viability with half-maximal effective concentrations (EC50) was evaluated after 72 h treatments. Antiproliferative and pro-apoptotic effects were also assessed for the most bioactive compounds (EC50 ≤ 50 µM). Oleocanthal (1) showed the strongest antiproliferative/cytotoxic activity in most cancer cell lines (EC50: 9−20 µM). The relative effectiveness of the six OOPs was: oleocanthal (1) > oleuropein aglycone (3a,b) > ligstroside aglycone (4a,b) > oleacein (2) > oleomissional (6a,b,c) > oleocanthalic acid (7). This is the first detailed study comparing the bioactivity of six OOPs in such a wide array of cancer cell lines, providing a reference for their relative antiproliferative/cytotoxic effect in the investigated cancers.

A Study on the Clustering of Extra Virgin Olive Oils Extracted from Cultivars Growing in Four Ionian Islands (Greece) by Multivariate Analysis of Their Phenolic Profile, Antioxidant Activity and Genetic Markers.

BACKGROUND: The phenolic fraction of extra virgin olive oil (EVOO) has disease preventive and health-promoting properties which are supported by numerous studies. As such, EVOO is defined as a functional food. The aim of the present study was to characterize the phenolic profile of olive oil from cultivars farmed in the Ionian Islands (Zakynthos, Kefalonia, Lefkada, and Kerkyra) and to investigate the association of phenols to antioxidant activity, which is central to its functionality. Furthermore, the study investigates whether multivariate analyses on the concentration of individual biophenolic compounds and genetic population diversity could classify the olive oil samples based on their geographic origin. METHODS: Phenols were determined in 103 samples from different Ionian Island tree populations by 1H nuclear magnetic resonance (NMR), and sample antioxidant activity was measured by their capacity to reduce the free radical 2,2-diphenyl-1-picrylhydrazyl) (DPPH). Genetic diversity was measured by estimating Nei's population genetic distance using 15 reproducible bands from random amplified polymorphic DNA (RAPD) genotyping. RESULTS: Principal component analysis (PCA) of the secoiridoid concentrations clustered samples according to cultivar. Clustering based on genetic distances is not concordant with phenolic clustering. A cultivar effect was also demonstrated in the association between the concentration of individual phenols with DPPH reducing activity. CONCLUSIONS: Taken together, the study shows that the olive oil phenolic content defines "cultivar-specific phenolic profiles" and that environmental factors other than agronomic conditions contribute more to phenotype variance than genetics.

Activation of specific bitter taste receptors by olive oil phenolics and secoiridoids.

Extra-virgin olive oil (EVOO) is a critical component of the Mediterranean diet, which has been found beneficial to human health. Bitterness is often positively associated with the presence of phenolic compounds in EVOO. There are twenty-five bitter taste receptors (TAS2Rs) in humans, each of which responds to specific bitter tastants. The identity of phenolic compounds and the bitter taste receptors they stimulate remain unknown. In this study, we isolated 12 phenolic and secoiridoid compounds from the olive fruit and the oil extracted from it, and tested their ability to stimulate bitter taste receptor activity, using a calcium mobilization functional assay. Our results showed that seven out of twelve studied compounds activated TAS2R8, and five of them activated TAS2R1, TAS2R8, and TAS2R14. The phenolic compounds oleuropein aglycon and ligstroside aglycon were the most potent bitter tastants in olive oil. TAS2R1 and TAS2R8 were the major bitter taste receptors activated most potently by these phenolic compounds. The results obtained here could be utilized to predict and control the bitterness of olive oil based on the concentration of specific bitter phenolics produced during the milling process of olives.

Silencing of Oleuropein ß-Glucosidase Abolishes the Biosynthetic Capacity of Secoiridoids in Olives.

Specialized metabolism is an evolutionary answer that fortifies plants against a wide spectrum of (a) biotic challenges. A plethora of diversified compounds can be found in the plant kingdom and often constitute the basis of human pharmacopeia. Olive trees (Olea europaea) produce an unusual type of secoiridoids known as oleosides with promising pharmaceutical activities. Here, we transiently silenced oleuropein ß-glucosidase (OeGLU), an enzyme engaged in the biosynthetic pathway of secoiridoids in the olive trees. Reduction of OeGLU transcripts resulted in the absence of both upstream and downstream secoiridoids in planta, revealing a regulatory loop mechanism that bypasses the flux of precursor compounds toward the branch of secoiridoid biosynthesis. Our findings highlight that OeGLU could serve as a molecular target to regulate the bioactive secoiridoids in olive oils.

A New Definition of the Term "High-Phenolic Olive Oil" Based on Large Scale Statistical Data of Greek Olive Oils Analyzed by qNMR.

In the last few years, a new term, "High-phenolic olive oil", has appeared in scientific literature and in the market. However, there is no available definition of that term regarding the concentration limits of the phenolic ingredients of olive oil. For this purpose, we performed a large-scale screening and statistical evaluation of 5764 olive oil samples from Greece coming from >30 varieties for an eleven-year period with precisely measured phenolic content by qNMR. Although there is a large variation among the different cultivars, the mean concentration of total phenolic content was 483 mg/kg. The maximum concentration recorded in Greece reached 4003 mg/kg. We also observed a statistically significant correlation of the phenolic content with the harvest period and we also identified varieties affording olive oils with higher phenolic content. In addition, we performed a study of phenolic content loss during usual storage and we found an average loss of 46% in 12 months. We propose that the term high-phenolic should be used for olive oils with phenolic content > 500 mg/kg that will be able to retain the health claim limit (250 mg/kg) for at least 12 months after bottling. The term exceptionally high phenolic olive oil should be used for olive oil with phenolic content > 1200 mg/kg (top 5%).

Spontaneous In Vitro and In Vivo Interaction of (-)-Oleocanthal with Glycine in Biological Fluids: Novel Pharmacokinetic Markers.

Since the first discovery of its ibuprofen-like anti-inflammatory activity in 2005, the olive phenolic (-)-oleocanthal gained great scientific interest and popularity due to its reported health benefits. (-)-Oleocanthal is a monophenolic secoiridoid exclusively occurring in extra-virgin olive oil (EVOO). While several groups have investigated oleocanthal pharmacokinetics (PK) and disposition, none was able to detect oleocanthal in biological fluids or identify its PK profile that is essential for translational research studies. Besides, oleocanthal could not be detected following its addition to any fluid containing amino acids or proteins such as plasma or culture media, which could be attributed to its unique structure with two highly reactive aldehyde groups. Here, we demonstrate that oleocanthal spontaneously reacts with amino acids, with high preferential reactivity to glycine compared to other amino acids or proteins, affording two products: an unusual glycine derivative with a tetrahydropyridinium skeleton that is named oleoglycine, and our collective data supported the plausible formation of tyrosol acetate as the second product. Extensive studies were performed to validate and confirm oleocanthal reactivity, which were followed by PK disposition studies in mice, as well as cell culture transport studies to determine the ability of the formed derivatives to cross physiological barriers such as the blood-brain barrier. To the best of our knowledge, we are showing for the first time that (-)-oleocanthal is biochemically transformed to novel products in amino acids/glycine-containing fluids, which were successfully monitored in vitro and in vivo, creating a completely new perspective to understand the well-documented bioactivities of oleocanthal in humans.

The Effect of Dietary Intervention With High-Oleocanthal and Oleacein Olive Oil in Patients With Early-Stage Chronic Lymphocytic Leukemia: A Pilot Randomized Trial.

AIM: Oleocanthal and oleacein (OC/OL) have important in vitro and in vivo antitumor properties; however, there is no data about their anticancer activity in humans. The aim of this pilot study was to test if patients at early stage of chronic lymphocytic leukemia (CLL) could adhere to and tolerate an intervention with high OC/OL extra virgin olive oil (EVOO) and if this intervention could lead to any changes in markers related to the disease. METHODS: A pilot dietary intervention (DI) was made in patients with CLL in Rai stages 0-II who did not follow any treatment (NCT04215367). In the first intervention (DI1), 20 CLL patients were included in a blind randomized study and were separated into two groups. One group (A) of 10 patients consumed 40 ml/day of high OC/OL-EVOO (416 mg/Kg OC and 284 mg/kg OL) for 3 months. A second group (B) of 10 patients consumed 40 ml/day of low OC/OL (82 mg/kg OC and 33 mg/kg OL) for 3 months. After a washout period of 9-12 months, a second intervention (DI2) only with High OC/OL-EVOO for 6 months was performed with 22 randomly selected patients (16 from the DI1 (8 from each group) and 6 new). Hematological, biochemical, and apoptotic markers were analyzed in the serum of the patients. In addition, cellular proliferation and apoptosis markers were studied in isolated proteins from peripheral blood mononuclear cells. RESULTS: The results of the DI1 showed beneficial effects on hematological and apoptotic markers only with High OC/OL-EVOO. During the DI2, a decrease in the white blood cell and lymphocyte count was observed (p ≤0.05), comparing 3 months before the intervention and 6 months after it. After 3 and 6 months of DI2, an increase (p ≤0.05) was observed in the apoptotic markers ccK18 and Apo1-Fas, and also in the cell cycle negative regulator p21, and also a decrease in the antiapoptotic protein Survivin, and in the cellular proliferation marker Cyclin D. CONCLUSIONS: This is the first clinical trial with High OC/OL-EVOO that indicates that it could be a promising dietary feature for the improvement of CLL inducing the apoptosis of their cancer cells and improving the metabolism of the patients. CLINICAL TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT04215367, identifier: NCT04215367.

Biotechnological Approaches on Two High CBD and CBG Cannabis sativa L. (Cannabaceae) Varieties: In Vitro Regeneration and Phytochemical Consistency Evaluation of Micropropagated Plants Using Quantitative 1H-NMR.

High cannabidiol (CBD) and cannabigerol (CBG) varieties of Cannabis sativa L., a species with medicinal properties, were regenerated in vitro. Explants of nodal segments including healthy axillary bud, after sterilization, were placed in Murashige-Skoog (MS) culture medium. The shoots formed after 30 days were subcultured in full- or half-strength MS medium supplemented with several concentrations of 6-benzyl-amino-purine (BA) or thidiazuron (TDZ). The highest average number and length of shoots was achieved when both full and half-strength MS media were supplemented with 4.0 µM BA. The presence of 4.0 µM TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 µM and 2.0, 4.0 µM respectively, displayed the maximum shooting frequency. The new shoots were transferred on the same media and were either self-rooted or after being enhanced with different concentrations of indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 µM IBA or 4.0 µM NAA resulted to the optimum rooting rates. The maximum average number and length of roots per shoot was observed when the culture media was supplemented with 4.0 µM IBA or NAA. Approximately 92% of the plantlets were successfully established and acclimatized in field. The consistency of the chemical profile of the acclimatized in vitro propagated clones was assessed using quantitative 1H-NMR high throughput screening. In each variety, analysis of the micropropagated plant in comparison with the mother plant showed no statistically significant differences (p ≤ 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.

Oleacein Attenuates the Pathogenesis of Experimental Autoimmune Encephalomyelitis through Both Antioxidant and Anti-Inflammatory Effects.

Oxidative stress and proinflammatory cytokines are factors affecting multiple sclerosis (MS) disease progression. Oleacein (OLE), an olive secoiridoid, possesses powerful antioxidant and anti-inflammatory activities, which suggests its potential application to treat neuroinflammatory disorders. Herein, we investigated the impact of OLE on the main clinic-pathological features of experimental autoimmune encephalomyelitis (EAE), an animal model for MS, including paralysis, demyelination, central nervous system (CNS) inflammation/oxidative stress and blood-brain barrier (BBB) breakdown. METHODS: Mice were immunized with the myelin oligodendrocyte glycoprotein peptide, MOG35-55, to induce EAE, and OLE was administrated from immunization day. Serum, optic nerve, spinal cord and cerebellum were collected to evaluate immunomodulatory activities at a systemic level, as well as within the CNS. Additionally, BV2 microglia and the retinal ganglion cell line RGC-5 were used to confirm the direct effect of OLE on CNS-resident cells. RESULTS: We show that OLE treatment effectively reduced clinical score and histological signs typical of EAE. Histological evaluation confirmed a decrease in leukocyte infiltration, demyelination, BBB disruption and superoxide anion accumulation in CNS tissues of OLE-treated EAE mice compared to untreated ones. OLE significantly decreased expression of proinflammatory cytokines (IL-13, TNFα, GM-CSF, MCP-1 and IL-1ß), while it increased the anti-inflammatory cytokine IL-10. Serum levels of anti-MOG35-55 antibodies were also lower in OLE-treated EAE mice. Further, OLE significantly diminished the presence of oxidative system parameters, while upregulated the ROS disruptor, Sestrin-3. Mechanistically, OLE prevented NLRP3 expression, phosphorylation of p65-NF-κB and reduced the synthesis of proinflammatory mediators induced by relevant inflammatory stimuli in BV2 cells. OLE did not affect viability or the phagocytic capabilities of BV2 microglia. In addition, apoptosis of RGC-5 induced by oxidative stressors was also prevented by OLE. CONCLUSION: Altogether, our results show that the antioxidant and anti-inflammatory OLE has neuroprotective effects in the CNS of EAE mice, pointing out this natural product as a candidate to consider for research on MS treatments.

A Randomized Clinical Trial of Greek High Phenolic Early Harvest Extra Virgin Olive Oil in Mild Cognitive Impairment: The MICOIL Pilot Study.

BACKGROUND: Extra virgin olive oil (EVOO) constitutes a natural compound with high protection over cognitive function. OBJECTIVE: To investigate for the first time the effect of Greek High Phenolic Early Harvest Extra Virgin Olive Oil (HP-EH-EVOO) versus Moderate Phenolic (MP-EVOO) and Mediterranean Diet (MeDi) in people with mild cognitive impairment (MCI). METHODS: We conducted a randomized prospective study so as to examine the HP-EH-EVOO and MP-EVOO versus MeDi in MCI. Genetic predisposition (APOEɛ4) to Alzheimer's disease (AD) was tested and an extensive neuropsychological battery was administered at baseline and after 12 months. Each participant was randomized and assigned one of three groups: 1) Group 1 received the HP-EH-EVOO (50 mL/day); 2) Group 2 received the MP-EVOO (50 mL/day), and 3) Group 3 received only the MeDi instructions. RESULTS: Better follow-up performance was found in Group 1 compared to Group 2 and Group 3 in the almost all cognitive domains. Moreover, Group 2 showed also significant improvement compared to Group 3 in ADAS-cog (p = 0.001) and MMSE (p = 0.05), whereas Group 3 exhibited worse or similar to baseline performance in almost all domains. In particular, Group 1 and Group 2 had better outcomes with regards to ADAS-cog (p = 0.003), Digit Span (p = 0.006), and Letter fluency (p = 0.003). Moreover, there was a significant difference (p = 0.001) in the presence of APOEɛ4 between the Groups 1 and 2 versus Group 3. CONCLUSION: Long-term intervention with HP-EH-EVOO or MP-EVOO was associated with significant improvement in cognitive function compared to MeDi, independent of the presence of APOEɛ4.

A Method for the Rapid Measurement of Alkylresorcinols in Flour, Bread and Related Products Based on 1H qNMR.

The main objectives of the current work were to investigate differences among flours from traditionally preserved Greek varieties of cereals, and especially those of wheat, and in parallel, to correlate those potential differences with the presence of bioactive natural ingredients. In this context, we developed a new, fast, and simple method for the measurement of total 5-alkylresorcinols in cereals and related foods by qNMR. Several types of flour (white or whole-grain) coming from wheat, i.e., Triticum dicoccum, T. monococcum, T. aestivum, T. durum and T. turgidum, corn, barley, rye and oat from a certified producer in Greece were used either as raw materials or for the production of bread, pasta and flakes. A small portion of the flour or the corresponding product was extracted with DMSO-d6. The liquid part was directly analyzed by NMR (400 MHz). The simplicity of the NMR spectrum of the total extract and the lack of overlapping peaks permitted the development of a high throughput quantitative method for the measurement of total bioactive alkylresorcinols in less than 15 min. Grains, whole grain flours and breads from old varieties of T. dicoccum and T.monococcum showed high contents of alkylresorcinols (455-1148 mg/Kg), while the same compounds were completely absent from white flour and the corresponding bread. The term high-phenolic flour is proposed to distinguish among flour types.

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.

High-Throughput 1H-Nuclear Magnetic Resonance-Based Screening for the Identification and Quantification of Heartwood Diterpenic Acids in Four Black Pine (Pinus nigra Arn.) Marginal Provenances in Greece.

A high-throughput quantitative Nuclear Magnetic Resonance 1H-NMR method was developed and applied to screen the quantity of the diterpenic resin acids in the heartwood of black pine, due to the renewed scientific interest in their medicinal properties and use in various diseases treatment. The 260 samples were taken from Pinus nigra clones, selected from four provenances of the Peloponnese (Greece), participating in a 35-year-old clonal seed orchard. Total resin acids per dry heartwood weight (dhw) varied greatly, ranging from 30.05 to 424.70 mg/gdhw (average 219.98 mg/gdhw). Abietic was the predominant acid (76.77 mg/gdhw), followed by palustric acid (47.94 mg/gdhw), neoabietic acid (39.34 mg/gdhw), and pimaric acid (22.54 mg/gdhw). Dehydroabietic acid was at moderate levels (11.69 mg/gdhw), while levopimaric, isopimaric, and sandaracopimaric acids were in lower concentrations. The resin acid fraction accounted for 72.33% of the total acetone extractives. Stilbenes were presented in significant quantities (19.70%). The resin acid content was composed mainly of the abietane type resin acids (83.56%). Peloponnesian Pinus nigra heartwood was found to be the richest source of resin acids identified to date and is considered the best natural source for the production of such bioactive extracts. The results indicate a high potential for effective selection and advanced breeding of pharmaceutical and high economic value bioactive substances from Pinus nigra clones.

Assessing Validity of Self-Reported Dietary Intake within a Mediterranean Diet Cluster Randomized Controlled Trial among US Firefighters.

Collecting dietary intake data is associated with challenges due to the subjective nature of self-administered instruments. Biomarkers may objectively estimate the consumption of specific dietary items or help assess compliance in dietary intervention studies. Our aim was to use a panel of plasma and urine biomarkers to assess the validity of self-reported dietary intake using a modified Mediterranean Diet Scale (mMDS) among firefighters participating in Feeding America's Bravest (FAB), an MD cluster-randomized controlled trial. In our nested biomarker pilot study, participants were randomly selected from both the MD intervention group (n = 24) and the control group (n = 24) after 12-months of dietary intervention. At baseline data collection for the pilot study (t = 12-months of FAB), participants in the control group crossed-over to receive the MD intervention (active intervention) for 6-months. Participants in the intervention group continued in a self-sustained continuation phase (SSP) of the intervention. Food frequency questionnaires (FFQ), 13-item-mMDS questionnaires, 40 plasma fatty acids, inflammatory biomarkers and urinary hydroxytyrosol and tyrosol were analyzed at both time points. Spearman's correlation, t-tests and linear regression coefficients were calculated using SAS software. Overall, the mMDS derived from the FFQ was highly correlated with the specific 13-domain-mMDS (r = 0.74). The concordance between the two questionnaires for low and high adherence to MD was high for all the participants in the parent trial (κ = 0.76). After 6 months of intervention in the pilot study, plasma saturated fatty acid decreased in both groups (active intervention: -1.3 ± 1.7; p = 0.002; SSP: -1.12 ± 1.90; p = 0.014) and oleic acid improved in the SSP (p = 0.013). Intake of olive oil was positively associated with plasma omega-3 (p = 0.004) and negatively with TNF-α (p < 0.001) at baseline. Choosing olive oil as a type of fat was also associated with higher levels of plasma omega-3 (p = 0.019) at baseline and lower TNF-α (p = 0.023) at follow up. Intake of red and processed meats were associated with lower serum omega-3 (p = 0.04) and fish consumption was associated with lower IL-6 at baseline (p = 0.022). The overall mMDS was associated with an increase in plasma omega-3 (p = 0.021). Good correlation was found between nutrient intake from the FFQ and the corresponding plasma biomarkers (omega-3, EPA and DHA). In this MD randomized controlled trial, some key plasma biomarkers were significantly associated with key MD diet components and the overall mMDS supporting the validity of the mMDS questionnaire as well as compliance with the intervention.