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Bryophyllum pinnatum (Lam) Pers. (Crassulaceae) is widely used in folk medicine as leaf juice, aqueous, or hydro-ethanolic extracts. It is also listed as a medicinal plant in several countries such as France and Brazil. The main reported constituents are flavone glycosides, especially those with the rare 3-O-α-l-arabinopyranosyl-(1 → 2)-α-l-rhamnopyranoside moiety. Despite several phytochemical screenings indicating the presence of cyanide derivatives or alkaloids, there are no reports of nitrogenous metabolite characterization from this plant species. Nevertheless, the occurrence and the type of such compounds are of particular interest, as they may account for some of the numerous biological activities and ethnomedicinal uses described for B. pinnatum and could be regarded as chemical/taxonomic markers. Consequently, a hydro-ethanolic extract of B. pinnatum was investigated by using UHPLC-HRMS/MS and the nitrile glucoside sarmentosin was detected for the first time within the genus Bryophyllum/Kalanchoe. Considering the wide use of B. pinnatum and its closely related species for health purposes, the target metabolite was isolated by a combination of centrifugal partition chromatography in elution/extrusion mode and MPLC in order to confirm its structure. A linear, selective, precise, fast, and reliable 1H NMR quantitation method was then developed and validated and may become a tool for easy quality assessment of the plant species. The amount of sarmentosin was determined as 2.07% of the examined sample. Sarmentosin was also detected in Kalanchoe laciniata, confirming the occurrence of this compound within the genus.

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.

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.

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 β-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.

Background Extra virgin olive oil is a food with a recognized health claim in the EU related to its phenolic content. Based on nuclear magnetic resonance (NMR) analysis, we observed for the first time that most high-phenolic olive oils also contain significant quantities of another potential beneficial ingredient, S-(E)-elenolide, which is a non-phenolic compound related to oleuropein or ligstroside. Elenolide had only been found in olive leaves and fruits as the Z isomer or had been synthesized and had been recognized as an antihypertensive agent. Results (E)-Elenolide was isolated from olive oil and its structure was elucidated and completely characterized for the first time using 1D and 2D NMR and gas chromatography-mass spectrometry. In addition, we developed a method of quantitative measurement based on qNMR. Investigation of 2120 olive oil samples showed that elenolide was present in the majority of samples, in quantities ranging from 0 to 2821 mg kg-1 . Although elenolic acid, which is a hydrated derivative of elenolide, had been reported as an olive oil ingredient, this is the first time that elenolide has proved to be transformed to elenolic acid after reaction with water. Finally, it was found that the quantity of elenolide in olive oil depends on the quantity of water remaining in the olive oil during storage. Conclusion S-(E)-Elenolide is a new important substance of olive oil and could be used as marker of high-quality oils with low water content. © 2019 Society of Chemical Industry.

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 &gt, 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 &gt, 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 &gt, 1200 mg/kg (top 5%).

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 &mu, M BA. The presence of 4.0 &mu, M TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 &mu, M and 2.0, 4.0 &mu, M 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 &alpha, naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 &mu, M IBA or 4.0 &mu, M 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 &mu, M 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 &le, 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.

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&rsquo, 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.

The phenolic profiles of extra virgin olive oils (EVOOs) may influence their cardiovascular benefits. In a randomized crossover of acute EVOO intake on platelet function, participants (n = 9) consumed 40 mL of EVOO weekly. EVOOs were matched for total phenolic content and were either tyrosol-poor with 1:2 oleacein/oleocanthal (D2i0.5), or 2:1 oleacein/oleocanthal (D2i2), or predominantly tyrosol (D2i0). Ibuprofen provided a platelet inhibition control. Blood was collected pre- and 2 h post-EVOO intake. D2i0.5 and D2i2 reduced 1 µg/mL collagen-stimulated maximum platelet aggregation (Pmax), with effects best correlated to oleocanthal intake (R = 0.56, P = 0.002). Total phenolic intake was independently correlated to eicosanoid production inhibition, suggesting that cyclooxygenase blockade was not responsible for the Pmax inhibition. Five participants exhibited >25% ΔPmax declines with D2i0.5 and D2i2 intake and plasma metabolomic profiles discriminated subjects by oil responsivity. Platelet responses to acute EVOO intake are associated with oil phenolic composition and may be influenced by diet.

During an investigation of the chemical profile of Greek figs (Ficus carica L.), several aqueous ethanol extracts (liquors) were prepared from dried Smyrna fig varieties cultivated in the two major fig-producing geographical areas in Greece: Peloponnese and Evia Island. The distinctive aroma observed among the prepared fig liquors led to the investigation of the odor profile of the different fig cultivars through HS-SPME coupled with GC/MS analysis, with focus on the factors that affect it before and during the preparation of the respective liquors.; Results: Significant variation in volatiles was noticed among all fig cultivars, as also between each fig cultivar pulp and the respective liquor. The observed diversity was a result of chemical reactions taking place in the ethanol matrix during the preparation of the liquor. The 'key' odor compound of dried fig aroma was found to be β-damascenone. Owing to its low detection threshold and minute quantity in fig cultivars, the presence of β-damascenone was furthermore confirmed through GC/MS/MS and GC/TOF-MS. β-Damascenone was identified in variable quantities among fig varieties and their liquors, predominating in Kalamon fig cultivar of Peloponnese region, while its amount was found to be dependent on the postharvest storage time and preservation process of dried figs.; Conclusion: Each of the studied fig cultivars and liquors showed a unique aroma profile, and the obtained results were used for the preparation of the first dried fig liqueur for potential commercial use with the highest content of β-damascenone. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

Mature ‘Chondrolia Chalkidikis’ olives were processed in an industrial olive oil mill equipped with a three-phase decanter. Water was added to the decanter at a 1:2 water-to-paste ratio. Olive mill waste water (ΟΜWW) was used to replace the added water at a rate of 50 or 100%. Following the final separation, the obtained oil was used for chemical analysis and sensory evaluation. All oils had similar acidity, peroxide and Κ values. OMWW-treated olive oils presented higher total phenolic content and higher antioxidant activity based on DPPH and oven tests, but lower chlorophyll and carotenoids content. However, there was no significant difference between the 50 and 100% replacement. The phenolic profile of the treated olive oils analyzed by quantitative 1Η NMR revealed more than twofold oleocanthal and oleacein as well as oleuropein and ligstroside aglycone contents than in the control. Sensory evaluation of treated oils also showed an enhancement of fruity, bitter and pungent attributes compared to the control.

Background: Recently published studies have demonstrated the strong anti-inflammatory properties of Scots pine (Pinus sylvestris L.) heartwood extracts, related to its stilbene content. In order to find alternative sources of Pinus heartwood extracts rich in stilbenes, a large number of samples were investigated, using a new developed high-throughput screening method based on quantitative nuclear magnetic resonance.; Results: The new method enabled us to measure the levels of pinosylvin, pinosylvin monomethyl ether and pinosylvin dimethyl ether in heartwood extracts in only 45 s per sample. The method was applied to 260 Pinus nigra trees originating from Peloponnese (southern Greece) from four different natural populations of the species. The results obtained showed that the total stilbenoids per dry heartwood weight varied greatly, ranging from 10.9 to 128.2 mg g-1drywood (average 59.92 ± 21.79 mg g-1drywood ). The major stilbene in all cases was pinosylvin monomethyl ether (40.32 ± 15.55 mg g-1drywood ), followed by pinosylvin (17.07±6.76 mg g-1drywood ) and pinosylvin dimethyl ether (2.54 ± 1.22 mg g-1drywood ). The highest stilbene content of P. nigra samples was found to be 6.3 times higher than the highest reported figure for P. sylvestris L.; Conclusion: Pinus nigra heartwood is the richest source of pinosylvin and pinosylvin monomethyl ether identified to date and can be considered the best natural resource for production of bioactive extracts. © 2016 Society of Chemical Industry.; © 2016 Society of Chemical Industry.

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.

Hypothesis/Purpose The aim of the present study was to evaluate in vivo the potential anti-ischemic and antiatheromatic activity of Chios Mastic gum, the resin of the trunk and branches of “ Pistacia lentiscus var. chia” , used since antiquity in traditional Greek medicine. The main compounds of mastic are triterpenes, possessing phytosterol-like structures. This led to the hypothesis that mastic and particularly its neutral fraction, enriched in phytosterol-like compounds, possess antiatheromatic activities. Methods Total Mastic Extract without Polymer (TMEWP) and the neutral mastic fraction (NMF) were administered orally for 6 weeks to normal fed and to cholesterol fed rabbits in the form of sunflower oil solution. All the animals were randomly divided into 6 groups, anesthetized and subjected to 30 min ischemia of the heart, followed by 3 h reperfusion: At the end of the experiment the area at risk and the infarct zone were determined with the aid of fluorescent particles and triphenyl tetrazolium chloride staining, and small segments of the ascending and descending aorta and the heart were taken for histologic examination. Blood samples were collected at different time points of ischemia and reperfusion, for malondialdehyde (MDA) evaluation as an index of lipid peroxidation, for total and LDL cholesterol determination and for evaluation of oxidized LDL. Results In the normal fed animals the NMF and the TMEWP reduced significantly the infarct size, while in the hypercholesterolemic rabbits both treatments were ineffective. Atherosclerosis was detected in all the animals fed cholesterol enriched diet in the form of subintimal accumulation of lipids and foamy macrophages. There was no detection of atherosclerosis in Groups treated with TMEWP and NMF, which both reduced the total cholesterol levels by 47 and 88% respectively, whilst had not effect on LDL oxidation. TMEWP and NMF reduced the MDA concentration in normal fed rabbits, but had no effect on MDA levels in cholesterol fed animals. TMEWP and NMPF reduce the infarct size in normal animals and possess significant antiatheromatic and hypolipidemic activities in rabbits fed cholesterol enriched diet.

BACKGROUND Olive oil contains compounds with interesting biological activities which are influenced by the cultivar, the geographic origin and other factors. The aims of this work were to (1) investigate these factors in Neb Jmel olive oil from various Tunisian origins; (2) determine the influence of geographic conditions on phenolic composition of Neb Jmel olive oil and consequently on the antioxidant compounds; and (3) verify whether oils could be discriminated based on geographical origin. RESULTS The characterisation of extra-virgin Neb Jmel olive oil produced in its original location has been conducted. Owing to the effect of the genotype and environmental, agronomic and technological factors on the chemical composition of olive oil and its quality, all studied olives were collected at the same season, and their oil obtained under the same processing technique. Many analyses were carried out to characterise the different olive oils: free acidity, peroxide value, fatty acid composition, Rancimat assay, pigments content and phenolic compounds by (1) H NMR. A recently developed method for the direct measurement of the oleocanthal and oleacein levels in olive oil by quantitative (1) H NMR was applied. The method was applied to the study of four Neb Jmel olive oils samples, and a broad variation of concentrations of all four secoiridoids was recorded. The concentration of each ranged from 55 to 529 mg kg(-1) and the sum of the four major secoiridoids (known as D3) ranged from 436 to 1063 mg kg(-1) . CONCLUSION The quantification of major phenolic compounds of olive oil by NMR indicated that environmental conditions influence the production of qualitative phenolic fractions. All these compounds can be used as base 'markers' to characterise and differentiate these olive oil on geographic origin. © 2016 Society of Chemical Industry.

Background: Malassezia yeasts produce bioactive indolic substances when grown on L‐tryptophan agar. A panel of these substances was tested against commensal and opportunistic fungi, the Minimum Inhibitory Concentration (MIC) was determined and the potential for in loco antifungal activity on the skin was assessed. Materials and Methods: Eight indoles were included (malassezin, pityriacitrin, indirubin, indolo[3,2‐b]carbazole, 6‐formylindolo[3,2‐b]carbazole, tryptanthrin, 6‐hydroxymethylindolo[3,2‐b]carbazole and 6‐methylindolo[3,2‐b]carbazole) and were tested against 40 fungal strains [yeasts: Malassezia spp.(N = 9); Cryptococcus spp.(N = 10); Candida spp.(N = 7); Yarrowia lipolytica(N = 1); Exophialla dermatitidis (N = 2); moulds: Aspergillus spp.(N = 7); Fusarium spp.(N = 2); Rhizopus oryzae(N = 2)]. The concentration of 5/8 of the tested indoles on diseased skin was calculated from published data. Kruskal‐Wallis and Mann‐Whitney U tests were employed for group susceptibility evaluation in 33 strains. Results: The MIC range was 0.125‐32 μg/mL, and the median log2MIC was four. Indirubin was the most potent antifungal agent and differed significantly from the others. The highest median MIC was found for FICZ. Malassezia with Candida strains were more susceptible compared to Cryptococcus and Aspergillus, and this inhibitory activity was predicted to be valid also on human skin. Conclusions: Malassezia yeasts produce indolic species that inhibit an array of clinically significant yeasts and moulds.

The investigation of olive oils stored for a period of 24 months under appropriate conditions (25 °C, dark place, and airtight container) led to the identification of a new major phenolic ingredient, which was named oleocanthalic acid. The structure of the new compound was elucidated using one- and two-dimensional nuclear magnetic resonance in combination with tandem mass spectrometry. The new compound is an oxidation product of oleocanthal and is found in fresh oils in very low concentrations. The concentration of oleocanthalic acid increased with storage time, while the oleocanthal concentration decreased. A similar increase of the oleocanthalic acid/oleocanthal ratio was achieved after exposure of olive oil to 60 °C for 14 days. Although the presence of an oxidized derivative of decarboxymethylated ligstroside aglycon had been reported, it is the first time that its structure is characterized. The isolated compound could induce the expression of amyloid-β major transport proteins as well as tight junctions expressed at the blood-brain barrier, suggesting that oleocanthalic acid could be beneficial against Alzheimer's disease.

Despite the evident influence of the cultivar on olive oil composition, few studies have been devoted to exploring the variability of phenols in a representative number of monovarietal olive oils. In this study, oil samples from 80 cultivars selected for their impact on worldwide oil production were analyzed to compare their phenolic composition by using a method based on LC–MS/MS. Secoiridoid derivatives were the most concentrated phenols in virgin olive oil, showing high variability that was significantly due to the cultivar. Multivariate analysis allowed discrimination between four groups of cultivars through their phenolic profiles: (i) richer in aglycon isomers of oleuropein and ligstroside; (ii) richer in oleocanthal and oleacein; (iii) richer in flavonoids; and (iv) oils with balanced but reduced phenolic concentrations. Additionally, correlation analysis showed no linkage among aglycon isomers and oleocanthal/oleacein, which can be explained by the enzymatic pathways involved in the metabolism of both oleuropein and ligstroside.

Four Dalmatian autochthonous olive cultivars (Buhavica, Drobnica, Lastovka and Oblica) were molecularly characterized by analyzing length variability of genomic DNA sequences encompassing 15 microsatellite repeats. Furthermore, several important parameters of olive oils were analyzed in relation to the harvest period. An analysis of major phenolics secoiridoids was done by qNMR, while the fatty acid profile of oils and squalene content was determined by GC–FID. Oxidative stability was evaluated by the Rancimat method and sensory evaluation was carried out by a trained professional panel. The results indicate that the effect of the harvest period on the phenolic profile of oils depends on the olive cultivar and is related to its genetic profile. Drobnica oil from the late harvest contained an extremely high concentration of oleocanthal + oleacein (966 mg/kg). The longest oxidative stability was achieved by Drobnica and Lastovka oils from the early harvest period (20.95 and 18.65 h). Squalene had no effect on the oil oxidative stability. This study shows that the content of phenolic secoiridoids depends mainly on the cultivar. In addition, some cultivars, such as Drobnica did not show significant change of phenolic secoiridoids content in relation to the harvest period.

Quinones are central intermediates in wine oxidation that can degrade the quality of wine by reactions with varietal thiols, such as 3-sulfanylhexanol, decreasing desirable aroma. Protection by wine preservatives (sulphur dioxide, glutathione, ascorbic acid and model tannin, phloroglucinol) was assessed by competitive sacrificial reactions with 4-methyl-1,2-benzoquinone, quantifying products and ratios by HPLC-UV-MS. Regioselectivity was assessed by product isolation and identification by NMR spectroscopy. Nucleophilic addition reactions compete with two electron reduction of quinones by sulphur dioxide or ascorbic acid, and both routes serve as effective quenching pathways, but minor secondary products from coupled redox reactions between the products and reactants are also observed. The wine preservatives were all highly reactive and thus all very protective against 3-sulfanylhexanol loss to the quinone, but showed only additive antioxidant effects. Confirmation of these reaction rates and pathways in wine is needed to assess the actual protective action of each tested preservative.

Nutritional value of extra virgin olive oil is associated with its complex chemical composition. The aim of this study was to determine phenolic secoiridoids in EVOOs from autochthonous Croatian cultivars (Drobnica, Krvavica, Lastovka, and Oblica) by qNMR, to determine simple phenolics by UPLC, as well as to analyse the fatty acid profile, the antioxidant activity and the oxidative stability of selected oils. This is the first study on chemical and biological characterization of selected autochthonous olives varieties. Drobnica EVOO contained the highest amount of total phenols and major secoiridoid derivatives (oleocanthal, oleacein, oleuropein aglycon, and ligstroside aglycon) compared to other oils. The antioxidant activity of Drobnica phenolics was very high by FRAP and copper-induced LDL oxidation assays, while the oxidative stability of Drobnica oil by Rancimat method was very long (23 h). Practical applications: This study represents the contribution to the research of chemical and biological potential of monovarietal extra virgin olive oil from Croatia. EVOOs from selected Croatian autochthonous cultivars had very high phenolic content that is related to high inhibitory rate of copper-induced oxidation of human LDL as well as the long oxidative stability. Drobnica EVOO showed very long oxidative stability. EFSA approved health claim on olive oil polyphenols (EU, 432/2012) and selected Croatian cultivars, especially Drobnica, are of interest due to its high phenolic content and strong biological potential.

A previously developed method for measurement of oleocanthal and oleacein in olive oil by quantitative (1)H NMR was expanded to include the measurement of the monoaldehydic forms of oleuropein and ligstroside aglycons. The method was validated and applied to the study of 340 monovarietal Greek and Californian olive oils from 23 varieties and for a 3-year period. A wide variation concerning the concentrations of all four secoiridoids was recorded. The concentration of each one ranged from nondetectable to 711 mg/kg and the sum of the four major secoiridoids (named as D3) ranged from nondetectable to 1534 mg/kg. Examination of the NMR profile of the olive oil extract before and after contact with normal or reversed stationary chromatography phase proved the artificial formation of the 5S,8S,9S aldehydic forms of oleuropein and ligstroside aglycon isomers during chromatography. Finally, methyl elenolate was identified for the first time as a minor constituent of olive oil.

Cochineal is one of the most well known organic red dyes. Dactylopius coccus Costa (Dactylopiidae) is a scale insect that is used as the source of the dye known as Mexican cochineal. Although cochineal is today a natural food colorant (E120) and although it has been used in art objects (textiles and paintings) for centuries, its exact chemical consistency is not well clarified except for carminic acid which is the major component and kermesic and flavokermesic acids. Several minor components (typically less than 5% of the colouring material) remained unknown or partially studied, although their presence has been reported in numerous analytical works related to art objects. Chemical investigation of the methanol extract of the dried insects, after subsequent HPLC chromatographic separations, led to the isolation and structure elucidation of six new anthraquinones, along with the known compounds carminic acid, kermesic acid and flavokermesic acid. The new compounds formerly described as DCII and DCIII, were found to be the 2-C-glucoside of flavokermesic acid and 4-aminocarminic acid, respectively, while DCIV and DCVII were found to be the α/β C-glucofuranosides of kermesic acid, and were studied as a mixture due to equilibrium. In addition, 3-O-glucoside of flavokermesic acid (DCOFK), and 3,4-dideoxycarminic acid (DDCA) were identified. The structures of the new compounds were elucidated on the basis of their NMR and MS data. Finally, the new compounds were detected in silk dyed with cochineal, lake pigment and, furthermore, in historical objects of the cultural heritage (icon and textile) using LC-DAD and LC-MS.

Malassezia yeasts are commensal microorganisms, which under insufficiently understood conditions can become pathogenic. We have previously shown that specific strains isolated from diseased human skin can preferentially produce agonists of the aryl hydrocarbon receptor (AhR), whose activation has been linked to certain skin diseases. Investigation of skin scale extracts from patients with Malassezia-associated diseases demonstrated 10- to 1,000-fold higher AhR-activating capacity than control skin extracts. Liquid chromatography-tandem mass spectrometry analysis of the patients' extracts revealed the presence of indirubin, 6-formylindolo[3,2-b]carbazole (FICZ), indolo[3,2-b]carbazole (ICZ), malassezin, and pityriacitrin. The same compounds were also identified in 9 out of 12 Malassezia species culture extracts tested, connecting their presence in skin scales with this yeast. Studying the activity of the Malassezia culture extracts and pure metabolites in HaCaT cells by reverse transcriptase real-time PCR revealed significant alterations in mRNA levels of the endogenous AhR-responsive genes Cyp1A1, Cyp1B1, and AhRR. Indirubin- and FICZ-activated AhR in HaCaT and human HepG2 cells with significantly higher, yet transient, potency as compared with the prototypical AhR ligand, dioxin. In loco synthesis of these highly potent AhR inducers by Malassezia yeasts could have a significant impact on skin homeostatic mechanisms and disease development.

The determination of mastic oil profile, with emphasis on its chiral characteristics, could serve as a method for detecting adulteration in products found in the market with a claim of mastic oil content aiming towards protecting it from counterfeiting. Furthermore the evaluation of the raw material is crucial, as the profile is potentially affected by factors as mastic origin and storage time. Thus 45 authentic mastic oil samples were analyzed by GC-MS employing a chiral column and content limits for all major constituents were determined. The chiral GC-MS analysis proved that selected concentration ratios between these constituents, namely those of (-)/(+)-α-pinene (≤1:100) and (-)-α-pinene/myrcene (1.9:100-11:100) could serve as markers for the determination of mastic oil authenticity. Employing this methodology, the analysis of 25 mastic oils contained in cosmetic and dietary products, as well as an artificial mastic oil sample, exhibited several differentiations that could indicate adulteration either with artificial essential oils or volatile compounds, or the use of aged mastic oil.

Oleocanthal and oleacein, two key secoiridoid derivatives present in virgin olive oil (VOO), are gaining clinical and nutritional interest thanks to their proved bioactivity; therefore, the determination of both phenols is a growing demanded application to increase the value of VOO. The main problem of previously reported liquid chromatography-based methods for oleocanthal and oleacein measurement is their interaction with water or other polar solvents such as methanol to promote the formation of hemiacetal or acetal derivatives. This interaction can occur during either sample extraction, basically liquid-liquid extraction, and/or chromatographic separation. The aim of this research was to evaluate the suitability of LC-MS/MS for absolute quantitation of oleocanthal and oleacein in VOO. For this purpose, both liquid-liquid extraction and chromatographic separation were studied as potential promoters of acetals and hemiacetals formation from oleocanthal and/or oleacein. The results showed that the use of methanol-water solutions for phenols extraction was not influential on the formation of these artifacts. Acetals and hemiacetals from oleocanthal and/or oleacein were only detected at very low concentrations when methanol gradients under acidic conditions were used for chromatographic separation. With this premise, a protocol based on extraction with acetonitrile and a reverse chromatographic gradient with methanol was established to quantify in absolute terms oleocanthal and oleacein in VOO samples. The resulting protocol was applied to three VOO samples characterized by high, medium, and low levels of these two phenols.

Data from limited dietary intervention trials suggest that the cardiovascular health benefit of EVOOs may increase as their phenolic antioxidants content increases. However, while EVOOs contain an ...

Background The aryl hydrocarbon receptor (AhR) is a nuclear receptor and transcriptional regulator with pleiotropic effects. The production of potent AhR ligands by Malassezia yeasts, such as indirubin, indolo[3,2-b]carbazole (ICZ), tryptanthrin and malassezin, has been associated with the pathogenesis of seborrhoeic dermatitis and pityriasis versicolor. Antigen-presenting cells in the skin can encounter microbes in the presence of these bioactive metabolites that could potentially modulate their function. Objectives To study the effects of the aforementioned naturally occurring ligands on AhR activation and Toll-like receptor (TLR)-induced maturation in human monocyte-derived dendritic cells (moDCs). Methods These indoles were screened for AhR activation capacity in moDCs employing CYP1A1 and CYP1B1 induction as read out and for their effects on the function of moDCs after TLR-ligand stimulation. Results Indirubin and ICZ were the most potent AhR ligands and were selected for subsequent experiments. Concurrent exposure of moDCs to indirubin or ICZ together with TLR agonists significantly augmented the AhR-mediated CYP1A1 and CYP1B1 gene expression. Additionally, mature DCs that were subsequently stimulated with AhR ligands showed increased AhR target gene expression. Moreover, these ligands limited TLR-induced phenotypic maturation (CD80, CD83, CD86, MHC II upregulation) of moDCs, reduced secretion of the inflammatory cytokines interleukin (IL)-6 and IL-12, and decreased their ability to induce alloreactive T-lymphocyte proliferation. Conclusions These results demonstrate that AhR agonists of yeast origin are able to inhibit moDC responses to TLR ligands and that moDCs can adapt through increased transcription of metabolizing enzymes such as CYP1A1 and CYP1B1.

The bis-indole alkaloid indirubin and its analogues bear a very interesting natural pharmacophore. They are recognized mainly as kinase inhibitors, but several other activities make them possible candidates for preclinical studies. Based on the previously reported activity of 7-bromoindirubin-3'-oxime and its derivatives, the synthesis of indirubins bearing a heterocyclic nitrogen atom at position 7 was carried out. Herein, we report the first synthesis of 7-azaindirubin-3'-oxime (12) as well as its antiproliferative activity against 57 cancer cell lines and its inhibitory activity against a series of kinases. 7-Azaindirubin (10) and its 3'-oxime derivative (12) showed reduced activity as kinase inhibitors in comparison with other known indirubin derivatives, but antiproliferative activity with a best GI(50) value of 0.77 microM.

Young fustic (Cotinus coggygria Scop.; Anacardiaceae) has been used as a dyestuff since antiquity. Phytochemical investigation of the methanol extract of the heartwood has led to the isolation and structure elucidation by nuclear magnetic resonance and mass spectrometry (MS) of 3',4',6-trihydroxyaurone (sulfuretin) and 3',4',7-trihydroxyflavonol (fisetin) as well as 3',4',7-trihydroxyflavanol (fustin), 3',4',5,7-tetrahydroxyflavonol (quercetin), 3',4',5,7-tetrahydroxyflavanol (taxifolin), 4',7-dihydroxyflavanol, 3',4',7-trihydroxyflavanone (butin), 4',7-dihydroxyflavanone (liquiritigenin), trans-2',3,4,4'-tetrahydroxychalcone (butein), 4',5,7-trihydroxyflavanone and trans-2',4,4'-trihydroxychalcone (isoliquiritigenin). The isolated compounds were used as reference materials for the development of a high-performance liquid chromatography-diode array detector-MS method, which was then applied to analyse (1) fresh silk samples dyed with young fustic, (2) dyed silk subjected to artificially accelerated light ageing and (3) historical silk micro-samples, extracted from ecclesiastical post-Byzantine garments (fifteenth to eighteenth century), which belong to monasteries of Mount Athos. Sulfuretin and fisetin, which are usually used as markers for the identification of the yellow dye and, for the first time, some of the aforementioned flavonoid components of young fustic were identified in the historical extracts. Furthermore, preliminary experiments suggested that although the amounts of the dye components decrease with light ageing, the relative ratio of fisetin and sulfuretin, after a first step of ageing, seems to be almost unaffected by such degradation processes raised by light. The effect of the latter on the morphology of the dyed silk fibres is briefly investigated by scanning electron microscopy.

Objectives We aimed to assess the intralumenal stability of oleuropein in human gastric and small intestinal contents. We additionally aimed to assess the stability characteristics of oleuropein in media simulating the intralumenal conditions. Methods The intralumenal stability of oleuropein was assessed in aspirates from the stomach and the upper small intestine of healthy volunteers collected under both fasted and fed state conditions and in media simulating the intralumenal environment. Key findings Oleuropein degraded in aspirates collected in the fasted state. When the initial concentration was about 50 μg/ml (close to expected intragastric concentration after single dose of commercially available products of oleuropein) the mean zero-order half-life of oleuropein in aspirates collected from the fasted small intestine was estimated to be 3.14 ± 0.08 h at 37°C (i.e. after oral administration in the fasted state, a substantial fraction of oleuropein degrades before reaching the intestinal mucosa). In contrast, oleuropein was stable in aspirates collected from the fed stomach; in small intestinal contents aspirated in the fed state the estimated zero-order degradation half-life was at least 12 h. Conclusions These data suggest that oleuropein should not have substantial intralumenal stability problems when administered in the fed state. Data collected in media simulating the intragastric and intraintestinal environment suggest that pH affects the stability of oleuropein only at low pH values (of about 2). At higher pHs degradation characteristics are at least partly affected by the presence of other scavengers of reactive oxygen species in the medium.

Twenty-nine analogs of indirubin, an isomer of indigo, have been synthesized to optimize its promising kinase inhibitory scaffold. These compounds being also pigmented, have been tested for their photoreactivity. Absorption maxima were between 485 nm and 560 nm. Addition of fetal calf serum induced fluorescence and time dependent absorption modifications. Appropriate illumination induced Reactive Oxygen Species (ROS) production for nineteen compounds out of twenty-nine. The relationship between fluorescence and ROS production is discussed. Six compounds showed an important toxicity on F98 cells, a murine glioma cell line. Three of these were found to be also phototoxic, as four other non-toxic compounds. All but onze phototoxic compounds were detected as ROS producers by in vitro tests. Photoreactivity assessment is important to anticipate adverse reactions for compounds that might be clinically developed. The experimental assay was found to be the only way to evaluate the photoreactivity of this family of compounds since no predictive criteria on structures could be found. Combining the vascular tumor growth inhibition induced by kinase inhibitors with the massive local blood flow arrest following photodynamic treatment may be an efficient anti-cancer strategy. These data could orientate further syntheses of either non-photoreactive compounds or compounds displaying both kinase inhibitory activity and strong phototoxicity.

Novel tetrasubstituted pyrazole derivatives bearing a nitro substituent on their A-phenol ring were synthesized and their binding affinity towards the estrogen receptor (ER) subtypes ERalpha and ERbeta was determined. Among compounds tested, the 2-nitrophenol derivative 5c was found to bind satisfactorily to both estrogen receptor subtypes (RBAalpha=5.17 and RBAbeta=3.27). In general, the introduction of a nitro group into the A ring of these compounds was found to benefit their ERbeta binding abilities.

The high polyphenol content of the wastewater is the major environmental problem caused by the olive mills. A pilot scale system for the treatment of the olive oil mills wastewater was developed aiming at the recovery of high added value-contained polyphenols and the reduction of the environmental problems. The treatment system consists of three main successive sections: The first one includes successive filtration stages aiming at the gradual reduction of the wastewater suspended solids up to a limit of 25 microm. The second section includes passing of the filtered wastewater through a series of adsorbent resins (XAD16 and XAD7HP) in order to achieve the de-odoring and decolorization of the wastewater and the removal/ recovery of the polyphenol and lactone content. The third section of the procedure includes the thermal evaporation and recovery of the organic solvents mixture, which has been used in the resin regeneration process, and finally the separation of the polyphenols and other organic substance contents using fast centrifuge partition chromatography. The final outcome of the whole procedure is (i) an odorless yellowish wastewater with a 99.99% reduced content in polyphenols and 98% reduced COD, (ii) an extract rich in polyphenols and lactones with high antioxidant activity and high added value, (iii) an extract containing the coloring substances of the olive fruit, and (iv) pure hydroxytyrosol.

A procedure, based on XAD-4 adsorption resin, which permits the obtainment of enriched polyphenolic extracts from Sesamum indicum perisperm (coat) has been developed. Chemical analysis of the obtained extracts led to the identification of 16 lignans. Among them, two new lignans, (+)-saminol and (+)-episesaminone-9-O-beta-D-sophoroside, have been isolated. Additionally, the relative stereochemistry of (-)-sesamolactol, previously reported as todolactol A epimer, has been unequivocally defined using X-ray crystallography. The structures of all new compounds were determined by spectroscopic methods, mainly by the concerted application of 1D and 2D NMR techniques (HMQC, HMBC, and NOESY) and mass spectroscopy.

A high performance liquid chromatography (HPLC) method combined with spectrophotometric UV‐Vis detection is developed for the separation and identification of seven indigoid coloring compounds: indigotin, which is commercially available, and indirubin, 6‐bromoindigotin, 6′‐bromoindirubin, 6‐bromoindirubin, 6,6′‐dibromoindigotin, and 6,6′‐dibromoindirubin, which are synthesized to be used as reference compounds in the HPLC analysis. The chromatographic method is employed for the identification of the blue/purple coloring compounds in samples extracted from four mollusks, which have been used for the production of Tyrian Purple since antiquity: Hexaplex trunculus, Murex brandaris, Nucella lapillus, and Thais haemastoma. The composition of the analyzed samples, with respect to the reference materials, is compared and discussed. All seven indigoids are identified in Hexaplex trunculus and some of them are identified in the other three purple extracts.

Indirubin, an isomer of indigo, is a reported inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) as well as an agonist of the aryl hydrocarbon receptor (AhR). Indirubin is the active ingredient of a traditional Chinese medicinal recipe used against chronic myelocytic leukemia. Numerous indirubin analogs have been synthesized to optimize this promising kinase inhibitor scaffold. We report here on the cellular effects of 7-bromoindirubin-3'-oxime (7BIO). In contrast to its 5-bromo- and 6-bromo- isomers, and to indirubin-3'-oxime, 7BIO has only a marginal inhibitory activity towards CDKs and GSK-3. Unexpectedly, 7BIO triggers a rapid cell death process distinct from apoptosis. 7-Bromoindirubin-3'-oxime induces the appearance of large pycnotic nuclei, without classical features of apoptosis such as chromatin condensation and nuclear fragmentation. 7-Bromoindirubin-3'-oxime-induced cell death is not accompanied by cytochrome c release neither by any measurable effector caspase activation. Furthermore, the death process is not altered either by the presence of Q-VD-OPh, a broad-spectrum caspase inhibitor, or the overexpression of Bcl-2 and Bcl-XL proteins. Neither AhR nor p53 is required during 7BIO-induced cell death. Thus, in contrast to previously described indirubins, 7BIO triggers the activation of non-apoptotic cell death, possibly through necroptosis or autophagy. Although their molecular targets remain to be identified, 7-substituted indirubins may constitute a new class of potential antitumor compounds that would retain their activity in cells refractory to apoptosis.

Eight new coumarins were isolated from the fruits of Seseli devenyense Simonkai. Their structures were established from NMR and mass data and their absolute configurations from chemical degradation correlation reactions. The new structures are the decanoic and dodecanoic esters of (+)-lomatin (3, 4), the decanoates of (+)-cis-khellactone at positions 4' (5) and 3' (6) as well as the 2'S epimer of 8-(2,3-dihydroxy-3-methylbutyl)-7-hydroxychromen-2-one (7) named devenyol, its two O-monoglucosides at positions 3' and 7 named devenyosides A (8) and B (9), and the corresponding 3'- and 7-O-diglucoside named devenyoside C (10). This plant is an interesting example of stereochemical diversity based on biodiversity given that other members of the Apiaceae family produce exclusively the 2'R epimers of compounds 7-9.

Two new alkaloids, (-)-cis-1,2-dihydroxy-1,2-dihydromedicosmine (3) and koniamborine (4), have been isolated from Boronella koniambiensis aerial parts. Their structures have been established from NMR and mass data. Koniamborine is a novel type of alkaloid, which derives from the pyrano[3,2-b]indole basic skeleton, described for the first time from nature. 6-Methoxy-1-methylisatin, also present in the plant material, can be considered biogenetically as a degradation product of the fused pyrone ring of 4.

The chemical compositions of the essential oils obtained from the aerial parts of Helichrysum kraussii and H. rugulosum were analysed by GC and GC–MS. From the 39 identified constituents, representing 85.1% and 92.9% of the two oils, respectively, β-caryophyllene (30.7%, 12.6%), α-pinene (12.1%,−), β-caryophyllene oxide (−, 8.8%), cis-α-bisabolene (−, 22.7%), β-bisabolene (−, 4.7%) and α-humulene (9.8%, −) were found to be the main components. Furthermore, the oils were tested against six Gram-positive or -negative bacteria and three pathogenic fungi. It was found that oils from both plants, and especially that of H. rugulosum, exhibited interesting antibacterial activity. Copyright © 2002 John Wiley & Sons, Ltd.

The chemical composition of the steam volatiles obtained from the inflorescences of six Euphorbia spp., E. acanthothamnos, E. apios, E. characias, E. dendroides, E. helioscopia and E. rigida, were analysed by GC and GC-MS. Sixty-nine components, representing 82–94% of the total composition for the six oils, were identified. The major components were found to be nonanal, germacrene, phytol, β-caryophyllene, heneicosane and heptacosane. Copyright © 2002 John Wiley & Sons, Ltd.

A practical synthetic scheme that incorporates an amide functionality into the rigid framework of (2 S ,3 R )-3-hydroxypipecolic acid to produce novel hydroxylated δ-lactam derivatives is reported. The reaction sequence includes the transformation of chiral furanylazide to a 2 S -hydroxymethyldihydro pyridone, which was reduced diasteroselectively, protected and oxidized to two corresponding δ-lactam derivatives. Asymmetric dihydroxylation and oxidation of the latter compounds afforded two chiral (2 R ,3 S )-3-hydroxypipecolic acid δ-lactam derivatives.

A new iridoid glucoside dimer, coelobillardin (1), was isolated from the aerial parts of Coelospermum billardieri. Its structure has been elucidated on the basis of MS and NMR data. From a biogenetic point of view, this compound, which includes a cyclobutane basic core, should be considered as a [2+2] dimerization product of (E)-10-(4-hydroxycinnamoyloxy)loganin, which is the major iridoid isolated from the plant.

The chemical composition of the essential oils of Achillea holosericea, Achillea taygetea, Achillea fraasii was determined by GC/MS analysis. Among the ninety-five assayed constituents, camphor, borneol and 1,8-cineol were found to be the major components. The in-vitro antimicrobial activity of these essential oils was evaluated against six bacteria indicating that the first is totally inactive, while the other two possess moderate to strong activities mainly against the Gram negative strains. The essential oil of A. fraasii was also active against the tested pathogenic fungi

A new glycoside of salicylic aldehyde, paeonicluside, was isolated from the roots of the Greek endemic species Paeonia clusii subsp. clusii and identified as α-ʟ-arabinopyranosyl- (1→6)-O-β-ᴅ-glucopyranoside of salicylic aldehyde (1). In addition, one characteristic monoterpene and two monoterpene glycosides were identified as paeoniflorigenone, paeoniflorin and benzoyl paeoniflorin, respectively. The structure of 1 was elucidated on the basis of its spectroscopic data and chemical correlation. It is the first time that a derivative of salicylic aldehyde is isolated from the well-studied Paeonia genus

Five new hexaoxygenated chalcones and one new chalcone photodimer were synthesized and their cytotoxicity against leukemia cell line L-1210 was studied. The three more active compounds were tested for their activity on the inhibition and promotion of tubulin assembly and it was found that these chalcones do not interfere with the tubulin-microtubule system at cytotoxic concentrations, and therefore operate by some different mechanism of action.

'Megaritiki' is an olive cultivar widely used in Greece for the production of low polyphenol olive oil and table olives. To investigate possible metabolic differentiation in comparison with other varieties, the composition of 'Megaritiki' olive fruits and wastewaters from the debittering procedure was studied. Moreover, the recovery of bioactive metabolites from wastewater using adsorption resin was studied to exploit this byproduct. Metabolites in fruits and wastewaters were monitored using NMR spectroscopy. The major constituents of wastewater were hydroxytyrosol-4-O-glucoside, 11-methyl-oleoside, hydroxytyrosol, and tyrosol but not oleuropein. Furthermore, wastewater afforded rengyoxide and rengyoside B, which are for the first time isolated from olives. The final edible olives, besides hydroxytyrosol and tyrosol, contained rengyoxide and cleroindicin C, which are the first isolated from the species, haleridone for the first time isolated from edible olives, and four metabolites, which are the first reported as natural products, megaritodilactone, megaritolactonic acid, methyl ester of megaritolactonic acid B, and megaritolactonol.

Homarine was isolated from nine edible species of marine molluscs belonging to classes Gastropoda, Bivalvia, and Cephalopoda. A thorough chromatographic, NMR and MS study provided evidence that homarine is a common and abundant metabolite of all these species. This study casts doubt on a previous assertion that 1,1′-dimethyl-[2,2′]-bipyridinium is a metabolite of the Bivalve Callista chione.