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We propose the cell culture approach to investigate oleuropein (1) biogenesis in Olea europaea L. We suggest employing olive callus cultures to identify the iridoidic precursor of oleuropein. In fact, we confirmed that callus cells from olive shoot explants are able to produce key secoiridoid as 1. To enable this approach, we synthesised and characterised deuterium-labelled iridoidic precursors belonging both to the loganin and the 8-epiloganin series. These iridoids are [7,8-(2)H2]-7-deoxy-8-epi-loganin (2(D)), [8,10-(2)H2]-8-epi-loganin (4(D)) and [7,8-(2)H2]-7-deoxy-loganin (3(D)).

Context: There is a need for the discovery of novel natural remedies to prevent and treat metabolic disorders such as hyperglycemia, type II non-insulin-dependent diabetes mellitus, and obesity. Several Achillea species have been utilized for centuries all around the world and are generally considered effective as hypoglycemic., Objective: Considering the ethnobotanical uses of Achillea genus, we evaluated the in vitro inhibitory activity of Achillea tenorii Grande (Asteraceae) extract on α-glucosidase, which is a valuable target to prevent and treat metabolic disorders. We also tested its antioxidant activity. Moreover, the phytochemical profile was discussed from a chemotaxonomic point of view., Materials and Methods: In vitro α-glucosidase inhibition of crude ethanolic extract obtained from the aerial parts was assayed as well as the in vitro antioxidant activity (ABTS, DPPH, and FRAP-FZ tests) was measured. The extract was characterized from a phytochemical point of view by means of spectroscopic analysis., Results: The extract results endowed with α-glucosidase inhibitory activity (IC50 32 µg/mL) with a particular mechanism of action definable as un-competitive, which differed from the mechanism observed for the best-known α-glucosidase inhibitor (acarbose and miglitol). In addition, a considerable antioxidant potential has been found for A. tenorii extract, which resulted mainly constituted by phenolic compounds such as caffeoylquinic acids and flavonoids., Discussion and Conclusions: These results suggest the potential of A. tenorii as a possible natural remedy to prevent and treat metabolic disorders of carbohydrates.

This work presents the first reported phytochemical study on the hydro-distilled essential oil from Achillea tenorii (Grande), collected in the protected area of Majella National Park (Italy). The composition of the essential oil was very different from those reported for the other species of Achillea nobilis group, being constituted mainly by oxygenated monoterpenes, among which ketones, alcohols and acetates compounds were the most representative. The marker compounds of A. nobilis group were not detected while the most abundant phytoconstituents were α-thujone (29.7%), trans-sabinol (18.6%) and trans-sabinyl acetate (15.7%), revealing a composition quite similar to that of Artemisia absinthium.

In this study, we evaluated, in the mouse, the effects of 20 mg/kg i.p. daily administration for 15 consecutive days of a blend of polyphenols, containing mostly oleuropein, extracted from the olive leaves (Olea europaea) on brain nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and on the expression of their receptors, TrkA, TrkB and p75. Polyphenols decreased the levels of reduced glutathione (GSH) and increased the levels of NGF and BDNF in the serum. In the brain, we found decreased levels of NGF and BDNF in the hippocampus and striatum but elevated levels of NGF in the olfactory lobes and hypothalamus and again BDNF potentiation in the olfactory lobes. No changes in TrkA, TrkB and p75 expression were observed. In conclusion, olive polyphenols may not only elicit an activation of the rodent olfactory system by increasing the levels of NGF and BDNF but also be stressing for the animal by reducing both the levels of hippocampal NGF/BDNF and serum GSH and increasing serum levels of NGF and BDNF.

In this study, we examined the composition regarding secondary metabolites of P. mugo Turra ssp. mugo growing in the protected area of Majella National Park, which is the southernmost station of the habitat of this species. Both the nonpolar and polar fractions were considered. In particular, the essential-oil composition showed a high variety of compounds, and 109 compounds were detected, and 101 were identified, among which abietane-type compounds have a taxonomic relevance. Abietanes were also isolated from the polar fraction, together with an acylated flavonol and a remarkably high amount of shikimic acid., (Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.)

The phytochemical study of the polar fraction of Bellardia trixago (L.) All. led to the isolation of eight iridoid glucosides. Five of these glucosides (aucubin (1), bartsioside (2), melampyroside (3), mussaenoside (4) and gardoside methyl ester (5)) were confirmed as they were previously isolated from this plant, and the remaining three known compounds (mussaenosidic acid (6), geniposidic acid (7) and 8-epiloganin (8)) were isolated here for the first time. Of particular interest were the presence of 7 and 8 due to two reasons: the first one because it is not accompanied with geniposide, the corresponding methyl ester, as in the case of 4 and 6, and the second one because it is the parent compound of iridoids characteristic of Orobanchaceae family. Also an alditol, D-mannitol (9), was recognised for the first time from this species.

Among HIV-1 reverse transcriptase (RT)-associated functions, DNA polymerase and Ribonuclease H (RNase H) are both essential for HIV replication and excellent targets for drug development. While all RT inhibitors approved for therapy target the DNA polymerase activity, there is the pressing need for new RT inhibitors possibly targeting the RNase H function. In the last 20 years, many natural substances have shown antiviral activity against HIV-1, but only a few against the RNase H function. In this study, we have tested the ethanolic extracts obtained by the Hypericum hircinum L. (Hypericaceae) growing in Sardinia (Italy) on the HIV-1 RT-associated RNase H function and found that they have inhibitory effects. Active extracts were fractionated up to obtain the main components that have been isolated, tested, and identified to be betulinic acid, shikimic acid, chlorogenic acid, quercetin, 5,7,3',5'-tetrahydroxyflavanone, and 5,7,3',5'-tetrahydroxyflavanone 7-O-glucoside. Betulinic acid and 5,7,3',5'-tetrahydroxyflavanone 7-O-glucoside were active on both RT-associated activities, and betulinic acid was also active on HIV-1 mutant RTs resistant to efavirenz. Overall, our results suggest that some of these compounds inhibit the HIV-1 RT binding to an allosteric site previously described for other natural compounds and are potential leads for further drug development of a single molecules having dual inhibitory activity., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Dihydroasparagusic acid (DHAA) is the reduced form of asparagusic acid, a sulfur-containing flavor component produced by Asparagus plants. In this work, DHAA was synthetically produced by modifying some published protocols, and the synthesized molecule was tested in several in vitro assays (DPPH, ABTS, FRAP-ferrozine, BCB, deoxyribose assays) to evaluate its radical scavenging activity. Results show that DHAA is endowed with a significant in vitro antioxidant activity, comparable to that of Trolox. DHAA was also evaluated for its inhibitory activity toward tyrosinase, an enzyme involved, among others, in melanogenesis and in browning processes of plant-derived foods. DHAA was shown to exert an inhibitory effect on tyrosinase activity, and the inhibitor kinetics, analyzed by a Lineweaver-Burk plot, exhibited a competitive mechanism. Taken together, these results suggest that DHAA may be considered as a potentially active molecule for use in various fields of application, such as pharmaceutical, cosmetics, agronomic and food.

Leaves of Olea europaea, cultivar Nocellara del Belice, were examined with respect to the medium-polar fraction, obtained by an ethyl acetate extraction of the whole extract. In the medium polar fraction, we isolated the two hydroxy-phenyl-ethyl alcohols (hydroxyl-tyrosol and tyrosol) that are the main component of olives. In addition, we isolated a flavonoidic compound, aromadendrine, a dihydroflavonol yet known but quite rare in nature. It is the first time that aromadendrine is isolated in O. europaea and we studied the aromadendrine biological activity. In particular, the ability of aromadendrine to reduce the inflammation induced in normal keratinocytes using an in vitro cell model was evaluated. The results of the present research indicate aromadendrine as a novel component in O. europaea with effective activity against skin inflammation.

The analysis of the polar fraction of Galeopsis angustifolia Ehrh. ex Hoffm. indicates that the main components are iridoids and flavonoids. Six compounds were identified: a new flavonoid, 3'-hydroxy-isoscutellarein 7-O-[6'″acetyl-β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranoside]; three iridoid glucosides: harpagide, acetyl harpagide and for the first time in this species, 8-epi-loganin; two known acetylated flavonoid glycosides: 3'-hydroxy-4'-O-methylisoscutellarein 7-O-[6'″acetyl-β-D-allopyranosyl-(1 → 2)-6″-acetyl-β-D-glucopyranoside], 3'-hydroxy-4'-O-methylisoscutellarein 7-O-[6'″acetyl-β-D-allopyranosyl-(1 → 2)-β-D-glucopyranoside]. Both flavonoids and iridoids are present in high amount; respectively, 16.7% and 4.5% of the crude extract.

Nocellara del Belice, a cultivated variety (cultivar) of olive tree (Olea europæa L.), was examined with respect to the medium-polar compounds present in the wastewaters of olive oil extraction at the end of 2007. Charcoal-polyamide chromatography of obtained wastewaters showed the presence of the chemotaxonomical markers of Olea europaea. In addition a new compound was isolated which resulted to be a lactone related to oleuropein aglycone. We propose the name of nocellaralactone (NOC). This compound is also present in the leaves and it appears to be structurally, probably biogenetically, related to jasminanhydride, a monoterpenoid previously isolated from Jasminum grandiflorum. NOC showed a significant in vitro anti-inflammatory activity.

In this study, we report the isolation and identification of several compounds present in the polar fraction of Stachys germanica L. subsp. salviifolia (Ten.) Gams, collected in the protected area of Majella National Park. In particular, we have isolated and identified harpagide, 7-β-hydroxy-harpagide, ajugol, 5-allosyloxy-aucubin, verbascoside and, for the first time in this genus, arbutin.

In this study, we report the isolation and identification of several compounds from Plantago sempervirens Crantz, collected in the protected area of Majella National Park. We examined the polar fraction, in particular the iridoidic one. Aucubin, caryoptoside, plantarenaloside and gardoside were isolated and identified. For the first time, in this species, 8-epiloganic acid was recognised. Also, verbascoside, a phenylethanoid glycoside, was recognised in this plant.

A series of bay-substituted perylene derivatives is reported as a new class of G-quadruplex ligands. The synthesized compounds have differing N-cyclic substituents on the bay area and differing side chains on the perylene major axis. ESI-MS and FRET measurements highlighted the strongest quadruplex binders in this series and those showing the highest quadruplex/duplex selectivity. Several biological assays were performed on these compounds, which showed that compound 5 (PPL3C) triggered a DNA damage response in transformed cells with the formation of telomeric foci containing phosphorylated γ-H2AX and 53BP1. This effect mainly occurred in replicating cells and was consistent with Pot1 dissociation. Compound 5 does not induce telomere damage in normal cells, which are unaffected by treatment with the compound, suggesting that this agent preferentially kills cancer cells. These results reinforce the notion that G-quadruplex binding compounds can act as broad inhibitors of telomere-related processes and have potential as selective antineoplastic drugs.

The flavonoid fraction of the aerial parts of Genista cadasonensis Valsecchi (Leguminosae), an endemic plant from Sardinia, was examined and compared with the flavonoid pattern already known in the Genista genus. This comparison evidenced the endemic nature of this species, showing the presence of a rare flavonoid, the 6-hydroxy-genistein. The antioxidant activity of dichloromethanic, ethanolic and acetonic total extracts of aerial parts was evaluated.

Secondary metabolite isolation and analysis of Plumbago europaea L. (Plumbaginaceae) is the aim of this work, which is included in a project of our groups on spontaneous and endemic Sardinian species. Plumbago europaea is the only specie of the genera that grows wild in Italy. Metabolites from leaves and roots of this specie were compared. Moreover, at present, literature on the phytochemical characterisation of P. europaea is poor. Plumbagin was isolated in both of the samples analysed, confirming its nature in the Plumbaginaceae family as chemotaxonomic marker of the Plumbaginae tribe. In this work, hydroplumbagin-4-O-glucoside and myricetin-3-O-rhamnoside are isolated for the first time in the genus.

The monoterpenoid fractions of three endemic Galium ssp. (Rubiaceae) from Sardinia Island were examined and compared with the iridoidic pattern yet known in Galium species. This comparison evidenced theirs endemic characters. In particular, in G.corsicum and in G. glaucophyllum loganic acid was isolated and identified for the first time in Galium genus. In G. schmidii a rare iridoid is present, 10-hydroxy-loganin, whose presence in this genus was evidenced only in G. mollugo and loganin isolated for the first time.

This work is to be included among an evaluation of analytical methods to be used for determination of olive oil quality. Studies on the diglycerides and phenols fraction have been conducted on some olive oils, obtained from cultivars grown in Calabria, during years 1997-2000. The analysis of obtained data indicates that there is a close relation between the content of phenols and diglycerides in virgin olive oil and that high content in phenols and 1,2-diglycerides is a characteristic of fresh olive oil.

Melissa officinalis L. (Lamiaceae) (lemon balm) is used in folk medicine for nervous complaints, lower abdominal disorders and, more recently, for treating Herpes simplex lesions. In this work the antiviral activity of a hydroalcoholic extract of lemon balm leaves against the Herpes simplex virus type 2 (HSV-2) was assessed by the cytopathic effect inhibition assay on Vero cells (ATCC CCL-81), in comparison with acyclovir. The cytotoxicity of the extract on Vero cells was previously tested by evaluating the cellular death and was confirmed by the Trypan blue test. Lemon balm showed to reduce the cytopathic effect of HSV-2 on Vero cells, in the range of non-toxic concentrations of 0.025-1 mg mL(-1) (with reference to the starting crude herbal material). The maximum inhibiting effect (60%) was obtained with 0.5 mg mL(-1). The viral binding assay showed that the extract does not prevent the entry of HSV-2 in the cells, thus suggesting a mechanism of action subsequent to the penetration of the virus in the cell. The extract was also chemically characterised by NMR and HPLC analysis; it showed to contain cinnamic acid-like compounds, mainly rosmarinic acid (4.1% w/w). Our experiments support the use of lemon balm for treating Herpes simplex lesions and encourage clinical trials on this medicinal plant.

Teucrium marum and T. subspinosum have been extracted with different polarity solvents, each crude extract was analysed with (1)H-NMR to compare the overall metabolic component. The polar extracts showed antioxidant activity in vitro. Verbascoside and arabinosyl-verbascoside were isolated and identified. Their concentrations were in connection to the antioxidant activity.

The phenyl propanoid fraction of Teucrium subspinosum Pourrette ex Willd. (Lamiaceae) from Baleari Island, was examined together with the same fraction of T. subspinosum from Sardinia. In T. subspinosum, besides verbascoside as main component, a new phenyl-propanoid-glycoside was isolated and identified by (1)H- and (13)C-NMR spectra analysis. Comparison with molecular composition of T. marum confirmed the differences between these species and evidenced their endemic character.

The iridoidic composition of Stachys glutinosa L. was examined in comparison with the results obtained in the phytochemical studies on S. corsica Pers. The presence of the known harpagide and acetyl-harpagide were showed together with that of a new di-glycosidic iridoid. The structure of this new compound, the 5-allosyloxy-aucubin, was demonstrated by comparison with the spectroscopical data of monomelittoside and of its 5-O-glucosyl-derivative, the melittoside. The presence of allose in Stachys genus seems to be a chemotaxonomical character.

The monoterpenoid composition of Verbascum conocarpum and Scrophularia trifoliata, both endemic plants of Sardinia, was examined. The main chemotaxonomic markers of Scrophulariaceae, the iridoids aucubin, verbascoside and catalpol, were isolated.

Two flavonoid glycosides containing acetylated allose, and two iridoid glucosides were obtained from a EtOH extract of Stachys corsica, an endemic plant of Sardinia and Corsica. All the compounds have previously been isolated from other species of Stachys.

In this work the iridoidic fraction of Teucrium marum from Sardinia, was examined. A new clerodane, teumarin B, was isolated and identified by 1H- and 13C-NMR spectra analysis. The endemic character of this plant was further confirmed. Teumarin B showed cytotoxic properties.