Phytochemical Analysis and Biological Activities of the Ethanolic Extract of Daphne sericea Vahl Flowering Aerial Parts Collected in Central Italy.

In this paper, the first phytochemical analysis of the ethanolic extract of Daphne sericea Vahl flowering aerial parts collected in Italy and its biological activities were reported. Eleven compounds were identified i.e., α-linolenic acid (1), tri-linoleoyl-sn-glycerol (2), pheophorbide a ethyl ester (3), pilloin (4), sinensetin (5), yuanhuanin (6), rutamontine (7), syringin (8), p-coumaric acid (9), p-anisic acid (10) and caffeic acid (11). To the best of our knowledge, compounds (1-4, 7-8 and 10) were isolated from D. sericea for the first time during this work, whereas sinensetin (5) represents a newly identified component of the entire Thymelaeaceae family. The extract was found to possess radical scavenging against both DPPH• and 2,2'-azino-bis(3-thylbenzothiazoline-6-sulfonic acid (ABTS•+) radicals, with at least a 40-fold higher potency against the latter. Moreover, chelating abilities against both ferrous and ferric ions have been highlighted, thus suggesting a possible indirect antioxidant power of the extract. Although the precise bioactive compounds remain to be discovered, the polyphenolic constituents, including phenolic acids, tannins and flavonoids, seem to contribute to the antioxidant power of the phytocomplex. In addition, the extract produced cytotoxic effects in MDA-MB-231 and U87-MG cancer cell lines, especially at the concentration of 625 µg/mL and after 48-72 h. Further studies are required to clarify the contribution of the identified compounds in the bioactivities of the extract and to support possible future applications.

Choline-chloride and betaine-based deep eutectic solvents for green extraction of nutraceutical compounds from spent coffee ground.

Coffee beans contain several compounds which have shown nutraceutial properties; among them chlorogenic acids (CGAs) are the most studied, have shown nutraceutical properties. These molecules are also present in the waste by-product known as spent coffee grounds (SCG). SCG are generated during the production of coffee drink, whereby roasted, ground coffee beans are heat or steam treated. A HPLC-PDA/ESI-MS method has been developed for the analysis of the most representative CGAs in SCG samples using a green extraction approach based on deep eutectic solvents (DESs). We examined fifteen different DESs based on choline chloride and betaine as hydrogen bond acceptor in combination with different hydrogen bond donors being seven alcohols, two organic acids, one sugar and urea. Fifteen CGAs were detected, identified and quantified for the evaluation of extraction efficiency. A betaine-based DES with triethylene glycol (molar ratio 1:2) was the most effective one at extracting CGAs from SCG also compared to a conventional organic solvent. Thirty percent of water added to DES and sample to solvent ratio 1:15 (g/mL) were selected as the best extraction conditions. The chromatographic method was validated studying LOD, LOQ, retention time and peak area precision, and linearity range. Retention time and peak area repeatability were determined obtaining RSDs values below 0.63 % and 8.51 %, respectively. Inter-day precision was determined obtaining RSDs value below 0.58 % and 8.80 % for retention time and peak area, respectively.

Application of deep eutectic solvents for the extraction of phenolic compounds from extra-virgin olive oil.

A HPLC-DAD/ESI-MS method has been developed and validated for the analysis of the most representative phenolic compounds in extra-virgin olive oil (EVOO) samples using a green extraction approach based on deep eutectic solvents (DESs) at room temperature. We examined ten DESs based on choline chloride and betaine in combination with different hydrogen bond donors comprising six alcohols, two organic acids, and one urea. Five phenolic compounds, belonging to the classes of secoiridoids and phenolic alcohols, were selected for the evaluation of extraction efficiency. A betaine-based DES with glycerol (molar ratio 1:2) was found to be the most effective for extracting phenolic compounds as compared to a conventional solvent. The optimization of the extraction method involved the study of the quantity of water to be added to the DES and evaluation of the sample-to-solvent ratio optimal condition. Thirty percent of water added to DES and sample to solvent ratio 1:1 (w/v) were selected as the best conditions. The chromatographic method was validated by studying LOD, LOQ, intraday and interday retention time precision, and linearity range. Recovery values obtained spiking seed oil sample aliquots with standard compounds at 5 and 100 µg/g concentration were in the range between 75.2% and 98.7%.

Anti-Candida Biofilm Activity of Pterostilbene or Crude Extract from Non-Fermented Grape Pomace Entrapped in Biopolymeric Nanoparticles.

Polymeric nanoparticle-based carriers are promising agents to deliver drugs to cells. Vitis vinifera phenolic compounds are known for their antifungal activity against Candida albicans. The aim of the present study was to investigate the antifungal activity of pterostilbene or crude extracts from non-fermented grape pomace, entrapped in poly(lactic-co-glycolic) acid nanoparticles (NPs), with diameters of 50 and 150 nm, on Candida biofilm. The fluorescent probe coumarin 6 was used to study the uptake of poly(lactic-co-glycolic)acid (PLGA) NPs in planktonic cells and biofilm. The green fluorescent signal of coumarin 6 was observed in Candida biofilm after 24 and 48 hours. Both pterostilbene and crude pomace extract entrapped in NPs exerted a significantly higher anti-biofilm activity compared to their free forms. The entrapment efficiency of both pterostilbene and crude pomace extract in PLGA NPs was ~90%. At 16 µg/mL, pterostilbene loaded in PLGA NPs reduced biofilm formation of 63% and reduced mature biofilm of 50%. Moreover, at 50 µg/mL, the pomace extract loaded in NPs reduced mature biofilm of 37%. These results strongly suggest that PLGA NPs are promising nanodevices for the delivery of antifungal drugs as the crude grape pomace extract, a by-product of white wine making.

Phytochemical profile of Euphorbia peplus L. collected in Central Italy and NMR semi-quantitative analysis of the diterpenoid fraction.

In this work, the phytochemical profile of the ethanolic extract of Euphorbia peplus L. collected in Central Italy, was reported. This specimen had never been studied before and the analysis was accomplished by means of Column Chromatography for the separation procedure and by means of NMR Spectroscopy and Mass Spectrometry for the identification step. In particular, fourteen compounds were evidenced belonging to five different classes of natural compounds i.e. triterpenoids (pentacyclic and saponin), peculiar diterpenoids (jatrophanes and pepluanes), flavonoids (flavonols), caffeoyl-quinic acids and rare disaccharides. In addition to this, a semi-quantitative analysis on the diterpenoid fraction, by means of NMR Spectroscopy, was also performed in order to provide the real quantities of these compounds in the same fraction and in the total extract. Due to the pronounced chemo variability observed in Euphorbia spp., the availability of a reliable and quick analytical technique, such as that reported in the present study, could be a useful tool in the standardization of plant materials to be used in pharmacological studies or for ethnomedicinal purposes. The technical details for both the general phytochemical analysis and the specific quantitative one, were inserted in this paper. Moreover, the chemotaxonomic and ethnopharmacological relevance of these compounds was also discussed.

Plasma Vitamin K1 Levels in Italian Patients Receiving Oral Anticoagulant Therapy for Mechanical Heart Prosthesis: A Case-Control Study.

BACKGROUND: Oral anticoagulant therapy (OAT) with a vitamin K antagonist (VKA) is the choice of treatment for preventing thromboembolism in patients with mechanical heart valve prosthesis (MHP). The percentage of time in the therapeutic range (TTR%) expresses the OAT quality. We planned a case-control study in order to determine vitamin K1 plasmatic concentrations in MHP patients and to correlate these with TTR%. MATERIALS AND METHODS: Of 756 MHP patients receiving OAT, 125 patients (61 younger than 65 years, and 64 older than 65 years) and 120 healthy blood donors, matched for sex and age, were enrolled in the study. All subjects completed a living questionnaire regarding diet, and underwent blood collection. Vegetable and fruit intake was categorized as optimal or suboptimal, and the high-performance liquid chromatography method was used to determine vitamin K1 levels. RESULTS: Neither the patients nor controls had been taking vitamin supplements prior to the start of the study. The median vitamin K1 level was 290 pg/mL in 72 controls with optimal intake, and 274 pg/mL in 48 controls with suboptimal intake, while the median vitamin K1 level in MHP patients with optimal intake was 409 pg/mL, significantly higher (p < 0.001) than the 133.5 pg/mL in patients with suboptimal intake. Vitamin K1 concentration in MHP patients appears to be linked to an age-related threshold: in patients younger than 65 years of age, the median vitamin K1 level was 431 pg/mL, significantly higher (p < 0.05) than the 290 pg/mL in patients older than 65 years of age. No clear relation was found between vitamin K1 levels and TTR% (Pearson = 0.14). However, patients with vitamin K1 >160 pg/mL showed a TTR% >60 %. Among patients younger than 65 years, subjects with vitamin K1 >160 pg/mL showed a median TTR of 66 %, this being significantly higher (p < 0.001) than the 46 % level shown by patients with vitamin K1 <160 pg/mL. CONCLUSIONS: Vitamin K1 concentrations in MHP patients seem to be related to both diet and age.

Constituents of Melittis melissophyllum subsp. albida.

In this paper, for the first time, we have reported the analysis of the polar fraction of Melittis melissophyllum subsp. albida, a species with ecological relevance which is also used in traditional medicine of Central-Southern Italy. Iridoid glucosides were mainly identified, together with verbascoside, an ubiquitous phenyl-ethanoid glycoside, with chemotaxonomic implications in Lamiales order. The majority of the isolated compounds is endowed with interesting bio- activities and may justify the traditional uses of this plant also from a chemical point of view. Several peculiarities were also recorded in the metabolic pattern of this subspecies, i.e. the presence of virginioside and geniposidic acid, two rare compounds in the Lamiaceae family. The presence of free cinnamic acid was an additional characteristic of this subspecies which showed a specific secondary metabolites content. These phytochemical peculiarities, together with the morphological differences showed by the subsp. albida in respect to the nominal species, may be a base for a reconsideration of the systematic of Melittis melissophyllum subsp. albida.

Olive oil mill wastewater treatment using a chemical and biological approach.

Olive oil mill wastewaters (OMW) are recalcitrant to biodegradation for their toxicity due to high values of chemical oxygen demand (COD), biological oxygen demand (BOD), and phenolic compounds. In the present study OMW, collected in southern Italy, were subjected first to a chemical oxidative procedure with FeCl3 and then to a biological treatment. The latter was performed in a pilot plant where mixed commercial selected bacteria, suitable for polyphenols and lipid degradation, were inoculated. The effect of treatments was assessed through COD removal, reduction of total phenols, and decrease of toxicity using primary consumers of the aquatic food chain (the rotifer Brachionus calyciflorus and the crustacean Daphnia magna). The results showed that the chemical oxidation was efficacious in reducing all parameters analyzed. A further decrease was found by combining chemical and biological treatments.

Environmental effects caused by olive mill wastewaters: toxicity comparison of low-molecular-weight phenol components.

Olive oil mill wastewaters (OMWs) show significant polluting properties due to their content of organic substances, and because of their high toxicity toward several biological systems. Wastewaters' toxicity has been attributed to their phenolic constituents. A chemical study of wastewaters from a Ligurian oil mill characterized phenolic products such as 1,2-dihydroxybenzene (catechol), derivatives of benzoic acid, phenylacetic acid, phenylethanol, and cinnamic acid. The OMWs were fractioned by ultrafiltration and reverse osmosis techniques and tested for toxicity on aquatic organisms from different trophic levels: the alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum); the rotifer Brachionus calyciflorus; and two crustaceans, the cladoceran Daphnia magna and the anostracan Thamnocephalus platyurus. The fraction most toxic to the test organisms was that from reverse osmosis containing compounds of low molecular weight (<350 Da), and this was especially due to the presence of catechol and hydroxytyrosol, the most abundant components of the fraction.