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Three water-soluble glucans (PELPS-A1, PELPS-A2, and PELPS-A3) purified from the hot water extract of the basidiomata of an edible mushroom Pleurotus eryngii var. elaeoselini by chromatography on DEAE-cellulose 32 and Sephadex G-100 column were found to consist of only D-glucose as monosaccharide constituent. Structural investigation was carried out by acid hydrolysis, periodate oxidation, and NMR experiments (1H-NMR, 13C-NMR, DQF-COSY, TOCSY, ROESY, HMQC, and HMBC). On the basis of these experiments, the structures of the repeating unit of the three isolated polysaccharides were established as follows: (1) PELPS-A1: {[→3)-α-D-Glcp-(1→]3→4)-α-D-Glcp-(1→2)-α-D-Glcp-(1→6)-α-D-Glcp-(1[→6)-β-D-Glcp-(1→]2}n 6 ↑ 1 α-D-Glcp (2) PELPS-A2: [→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→6)-β-D-Glcp-(1→]n 3 ↑ 1 β-D-Glcp (3) PELPS-A3: [→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→]n The dried basidiomata of P. eryngii var. elaoselini were tested for their antioxidant activity by DPPH and hydroxyl radical scavenging assays. The crude extract has shown a SC50 of 1.4 mg/mL for DPPH test while a SC50 of 5.7 mg/mL was observed for hydroxyl radical scavenging activity test. The antioxidant activity of PELPS-A1, PELPS-A2, and PELPS-A3, evaluated as hydroxyl radical scavenging activity, was similar and significant, suggesting their use as antioxidants.

In 2000, a new variety of P. eryngii associated with the root residues of Elaeoselinum asclepium (L.) Bertol. subsp. asclepium (Apiaceae) was described. P. eryngii var. elaeoselini Venturella, Zervakis & La Rocca is an entirely white edible mushroom distributed along the Italian territory where it grows, in autumn and spring ,from 0 to 1200 m a.s.l.. The variety was also found in Spain, on different host plants, i.e. Thapsia villosa L. and Elaeoselinum gummiferum (Desf.). Tutin. Further investigation highlighted the presence of the variety also in France, Switzerland, Malta, Slovenia, Ukraine and Romania on Laserpitium latifolium L. Preliminary investigation carried out on basidiomata of P. eryngii var. elaeoselini collected in Sicily (southern Italy) highlighted a low caloric content and interesting amount of vitamins and mineral salts. In more recent times, extracts of P. eryngii var. elaeoselini, were tested for their in vitro growth inhibitory activity against a group of bacterial reference strains of medical relevance: Staphylococcus aureus ATCC 25923, S. epidermidis RP62A, Pseudomonas aeruginosa ATCC 15442, and Escherichia coli ATCC10536. The extracts of P. eryngii var. elaeoselini inhibited the tested microorganisms in varying degrees. In this study the structural characterization of the aqueous extract obtained from P. eryngii var. elaeoselini is investigated. Different water-soluble glucans were obtained from the fruiting bodies by hot water extraction, ethanol precipitation, dialysis, DEAE cellulose ion exchange chromatography and Sephadex G-100 gel filtration. On the basis of total hydrolysis, methylation analysis and NMR experiments (1H, 13C, DQF-COSY, TOCSY, ROESY, HSQC and HMBC) different polysaccharide fractions were characterized and the structures of the repeating units of the glucan were determined. Monosaccharide composition analysis revealed that the water soluble polysaccharide fraction mainly consisted of glucose (96.3%), with a small amount of mannose (3.7%). The sequence of glycosyl residues in the polysaccharide fractions was confirmed from HMBC experiments. Long-range 13C-1H correlations were obtained from the HMBC spectrum and the cross peaks of both anomeric protons and carbons of each of the sugar moieties were examined and both inter- and intraresidual connectivities were observed. The dry fruiting bodies of P. eryngii var. elaeoselini have shown a significative antioxidant activity with the DPPH test, while the purified polysaccharides have confirmed this activity with the hydroxyl radical scavenging activity test.

A preliminary biological investigation of the dry basidiomata of strain C-142-c of Pleurotus eryngii has shown significant antioxidant activity. Two different polysaccharides (PEPS-A1 and PEPS-A2) were isolated from the cultivated edible mushroom, P. eryngii C-142-c strain. Based on acid hydrolysis, methylation analysis, and nuclear magnetic resonance experiments (1H, 13C, distortionless enhancement by polarization transfer, double quantum filtered correlation spectroscopy, total correlation spectroscopy, nuclear Overhauser effect spectroscopy, heteronuclear singlequantum correlation spectroscopy, and heteronuclear multiple-bond correlation spectroscopy), the structures of the repeating unit of PEPS-A1 and PEPS-A2 were established as follows: (1) PEPS-A1 (a-glucan): [→6)-a-D-Glcp-(1→6)- a-D-Glcp-(1→]n; and (2) PEPS-A2 (b-glucan): [→6)-b-D-Glcp-(1→6)-b-D-Glcp-(1→]n. The antioxidant activity of PEPS-A1 and PEPS-A2 was evaluated as hydroxyl radical scavenging activity. PEPS-A1 and PEPS-A2 showed SC50 values of 400 µg/mL and 122 µg/mL, respectively, suggesting their possible use as a dietary supplement in functional foods. The polysaccharides were tested for their activity on cell viability using a colorectal adenocarcinoma cell line (HT29). Both polysaccharides affected cell viability after 48 and 72 hours of treatment, inducing the death of 50% of HT-29 cells between 0.25 and 1 µg/mL and between 0.5 and 1 µg/mL, respectively, for PEPS-A1 and PEPS-A2. These results are promising for future applications of these mushroom-derived polysaccharides as antioxidants and antitumor agents.

The mycochemical composition and structural characterization of the whole aqueous and methanolic extracts from Pleurotus eryngii var. eryngii are investigated. Two water-soluble glucans were obtained from the fruiting bodies by hot water extraction, ethanol precipitation, dialysis and Sephadex G-200 gel filtration. On the basis of total hydrolysis, methylation analysis, and NMR studies (1H, 13C, DQF-COSY, TOCSY, HSQC and HMBC) two fractions PEPS-1 and PEPS-2 were characterized and the structure of the repeating unit of the glucan were determined as linear (1→6)--glucan and (1→6)--glucan, respectively. The methanolic extract of Pleurotus eryngii var. eryngii was also investigated and a complex mixture of triglycerides together with ergosterol, ergosterol-3-O--linoleate were identified. The structures were established on the basis of spectroscopic data and chemical reactions. The dry fruiting bodies of Pleurotus eryngii var. eryngii have shown a significative antioxidant and antitumour activity.

Among basidiomycete molecules, cell wall polysaccharides have been recognized as a major class of bioactive constituents. [1] They are safe molecules and they have a wide spectrum of biological activities, such as immunostimulatory and antioxidant, therefore they possess a prominent role in health benefits coming from mushroom consumption. These properties make mushroom polysaccharides potential candidates for nutraceutical applications and bioactive ingredients production. [2] Fractionation of the hot aqueous extract of Cortinarius caperatus led to isolation of two fractions characterized by spectroscopic analyses (1H-NMR, 13C-NMR, DEPT, 1H-1H COSY, DQCOSY, TOCSY, HSQC, HMBC and HMQC), mass spectrometry (EI-MS, ESI-MS), infrared spectroscopy (FT-IR), chemical reactions of hydrolysis and derivatization followed by GC and HPLC analyses. [3] This mycochemical study revealed a water-soluble fraction characterized as a β-(16)-D-glucan, whose presence inside C. caperatus has never, to the best of our knowledge, been reported before. Moreover, a water insoluble fraction purified has been characterized as a branched , (1→6) glucan which structure is assumed to be: [→6)-β-D-Glcp(1→6)]4-α-D-Glcp(1→4)-β-D-Glcp(1→ 6 ↑ 1 α-D-Glcp The antioxidant activity of the soluble polysaccharide fraction has been evaluated as radical-scavenging activity with the DPPH test, the β-(16)-D-glucan showed significative antioxidant activity.

The term nutraceutical can be applied to any food or active principle able to determine positive effects on health, including any preventive effect and treatment of diseases. One of the areas of most interest involves the use of nutraceuticals in cardiovascular prevention. The ubiquinol (UBI), also considered the CoQ10 of next generation, is a raw material for food supplements with antioxidant activity; it plays a key role in energy production and in increasing body resistance1. In recent years nutraceuticals constituted by branched chain amino acids (Branch Chain Amino Acids, BCCA) have found increasing use as anti-fatigue active elements1. The muscle can directly use the BCAA for protein synthesis (plastic function) can also for gluconeogenesis (energy function). The main objective of this work is to increase the water solubility and the ubiquinol stability through chemical modifications with polyethylene glycol (PEG) using spacer arms, consisting of specific amino acids, alanine, valine, isoleucine and leucine. The polyethylene glycol (PEG) is a synthetic polymer approved by the Food and Drug Administration, highly water-soluble, biocompatible, non-teratogenic, non-immunogenic, antigenic and non-toxic, easily eliminated from the human body and widely used in drug delivery system2. Conjugates PEG-BCAA-UBI should have a possible synergistic action in energy production in muscles. The synergistic action could be extremely interesting when applied, particularly, to the heart muscle. The carboxylate polymer is the starting building block for the preparation of mPEG-UBI, mPEG-Ala-UBI and mPEG-BCAA-UBI. Pharmacokinetic studies show that the adducts deliver the corresponding beneficals amino acids together with ubiquinol giving a good antioxidant action.

a-Methylenelactones have received much attention due to the biological activity exhibited by natural products containing this structural moiety [1]. Cytotoxic, anti-inflammatory, phytotoxic, allergenic and antimicrobial properties are shown not only by highly functionalized, complex sesquiterpene lactones but also simple representatives were studied for their biological effects. Besides, compared to normal human tissues, many common human cancers, express high levels of fatty acid synthase (FAS), the primary enzyme responsible for the synthesis of fatty acids. This differential expression of FAS between normal tissues and cancer has led to the notion that FAS is a target for anticancer drug development [2]. A new series of a-methylene-g.butyrolactones has been synthesized with an alkylidene moiety in 3 position and a substution in 5 position. All the compounds were tested against PANC-1, HCT-116, Lovo, MCF-7 and MDA-231-MB tumor cell lines by MTT assay to different concentrations of the above compounds (4.1x10-7-3.0x10-4 ) after a 48 h exposition. The compound with R=H and R'= 3',5'- dimethoxyphenyl exhibited a pharmacological interesting cell toxicity profile in all cell lines with EC50 values similar to the reference compound cerulenine. References 1) F. Cateni et al. , European Journal of Medicinal Chemistry 41, (2006), 192-200. 2) F.P. Kuhajda et al. , Proc. Natl. Acad. Sci. USA 7, (2000), 3450-4.

The commercial pumpkin seed oil (Cucurbita pepo L.) has been investigated. The oil is a common product in Slovenia , Hungary and Austria and is considered a preventing agent for different pathologies, particularly for prostate diseases. These properties are related to the high content in carotenoids and liposoluble vitamins. In the present paper the carotenoids (lutein and zeaxantine), vitamin E (-and -tocopherol) and fatty acids content has been investigated. Furthermore, the composition of the volatile fraction due to roasting process is investigated. The results of this study show that the aromatic profile obtained from the commercial analyzed samples is directly related to the intensity of the roasting process of the crushed pumpkin seeds: the temperature of the roasting process plays a crucial role on the concentration of volatiles substances, originated from Strecker degradation, lipid peroxidation and Maillard reaction. The findings suggest that high temperature roasting processes lead to the production of an oil with intense aromatic characteristics, while mild conditions, generally employed to obtain an oil with professed therapeutic characteristics, would lead to a product with minor characteristics pumpkin seed oil aroma. The nutraceutic properties of the product are confirmed by the high content in -and -tocopherol and carotenoids.

The commercial oil of the seeds of pumpkin (Cucurbita pepo L.) has been investigated. The oil is a common product in Slovenia, Hungary and Austria and is considered for its health promoting properties. Therefore, the pumpkin seed oil is considered a preventing agent for different pathologies, particularly for prostate diseases. These properties are related to the carotenoids and liposoluble vitamins high content. The carotenoids (lutein and zeaxantine), vitamin E (α-tocopherol) and fatty acids content has been investigated. Furthermore, the composition of the volatile fraction due to roasting process has been investigated. The preliminary extraction of volatile compounds was obtained by dynamic headspace, while the analyses were performed by gas chromatography-mass spectrometry. The results of this study show that the aromatic profile obtained from the commercial analyzed samples is directly related to the intensity of the roasting process of the crushed pumpkin seeds: the temperature of the roasting process plays a crucial role on the concentration of volatiles substances, originated from Strecker degradation, lipid peroxidation and Maillard reaction. Therefore, high temperature roasting processes lead to the production of an oil with intense aromatic characteristics, while mild conditions, generally employed to obtain an oil with professed therapeutic characteristics, would lead to a product with minor characteristics pumpkin seed oil aroma. The nutraceutic properties of the product are confirmed by the high content in a-tocoferol and carotenoids: in particular, the content in lutein and zeaxanthin is one order of magnitude higher to the virgin olive oil.

Amanita fungi are the main lethal toadstool among the 1000 poisonous mushrooms known in the world. Thrombin is the key serine proteinase of the coagulation cascade and therefore a suitable target for inhibition of blood coagulation. An extract of Amanita virosa considerably inhibited thrombin (48%) and showed no inhibitory activity on trypsin. On the basis of inhibition selectivity between thrombin and trypsin and potency of thrombin inhibition, Amanita virosa constituted a good starting material for isolation of further compounds that are active against thrombin. In a preliminary study, 95 selected mushroom species have been screened in order to find novel specific non-peptidic thrombin inhibitors. The extract of Amanita virosa considerably inhibited thrombin (48% at concentration of 120 μg/mL). A bioassay oriented fractionation of the extract of Amanita virosa has led to the isolation of active compounds. On the basis of spectroscopic data, chemical reactions and GCMS analysis, complex mixtures of triglycerides, monoacylglycerols, free fatty acids and ergosterol have been isolated and identified.

Photodynamic therapy (PDT) is a non-invasive therapeutic modality used in a various number of diseases and cancer. It involves the systemic or topic administration of a photosensitizer, followed by irradiation with light. The activated photosensitizer converts oxygen to singlet oxygen and/or reactive oxygen species (ROS) which lead to cell death and tissue necrosis. One aim of PDT research is the discovery of new photosensitizers possessing minimal dark cytotoxicity, high photodynamic properties, improved pharmacokinetics, preferential retention in diseased instead of healthy tissues, chemical stability and a good cellular uptake .We recently focused our efforts on pheophorbide a (Pba), a chlorophyll derivative. Pba is characterized by a stronger absorption between 650-700 nm, in the tissuepenetrating wavelength range. For in vivo applications the capacity of the photosensitiser to reach in the diseased tissues becomes critical, in particular when a large peritoneal area is interested as occurring in carcinomatosis and sarcomatosis. To improve the pharmacokinetic and the activity of the photosensitizer we conjugated Pba to polyethylene glycol (PEG). In vivo the pharmacokinetic analysis performed on living female C57/BL6 mice bearing a subcutaneous melanoma mass, showed that injected mPEG-Pba distributes all over the body, with an higher uptake in the tumor respect to free Pba. Moreover, preliminary data suggest that PEG-Pba in mice bearing B78-H1 amelanotic melanoma reduces the tumor growth after light activation in comparison with Pba.

The search for drugs to treat cystic fibrosis (CF) has to take into account the large number of mutations affecting the gene and the variety of molecular mechanism through which these mutations cause the functional defect. For instance, deletion of phenylalanine at position 508 (DF508), the most frequent mutation (occurring in more than 50-70 % of patients), causes both a severe defect of CFTR protein processing (trafficking defect) and a decrease of its channel activity (gating defect). Recently, we have found that some 1,4-dihydropyridine (DHP) derivatives used to treat hypertension are able to stimulate the activity of DF508- and G551D-CFTR mutants. As DHPs present only a type of activity facilitating CFTR gating, they must be defined as Potentiators. Antihypertensive DHPs act by blocking L-type voltage-dependent Ca++ channels and therefore cause the relaxation of arterial smooth muscle cells. It has been shown that mutant CFTR is activated by DHPs through a mechanism not involving the modulation of Ca++ channels, but the direct interaction with the CFTR protein itself. These findings prompted us to synthesize DHP derivatives in order to study structure-activity relationships on the modulation of the CFTR-dependent anion transport. Now, we are presenting a series of new DHPs bearing in position 4 a furan-2-yl substituent. DHPs were tested on Fisher rat thyroid cells co-expressing DF508-CFTR mutation and a halide-sensitive yellow fluorescent protein. Cells were incubated at 27 °C to lead the mutant CFTR into the plasma membrane, so rescuing the protein from the endoplasmic reticulum. The tested compounds presented a significant degree of potency against rescued DF508-CFTR. The best activity was found when R = CH2C6H5, R1= H, R2=H, R3= C6H5. Studies of Molecular Modeling on DHPs are in progress: our preliminary data show that DHPs lay into a channel near to the ATP pocket in NBD1 of CFTR (Figure 2): the presence of Potentiators in this site could make more difficult the hydrolysis of ATP, having as a consequence a longer opening time of the CFTR channel.

Plantago major has been used in traditional medicine for the treatment of various diseases. In order to evaluate the quality of P. major, a simple, rapid and accurate high-performance liquid chromatography (HPLC) method was developed for the assessment of the two bioactive triterpenic acids: oleanolic acid (OA) and ursolic acid (UA). A LiChrosorb octadecylsilyl silica (ODS) column with methanol, tetrahydrofuran and aqueous acetic acid as mobile phase and detection at 220 nm were employed for HPLC analysis. The method is precise with relative standard deviations for these two constituents that ranged between 0.5–1.1% (intraday) and 0.8–1.8% (interday). The contents of these two phytochemicals in the leaves of P. major growing in Friuli Venezia Giulia Region were determined in order to establish an effective and reproducible method.

Poly(ethylene glycol) esters have been proposed as a potential oral-controlled drug delivery system for dexamethasone (DXM). The drug was covalently attached to monomethoxypolyethylene glycol amine (mPEG-NH2) (Mw = 10,000 Da) using a succinate linker. A new PEG-based prodrug system was designed using ester derivatives. The drug-polymer conjugate (mPEG-DXM) was freely water soluble at room temperature. First of all, the adduct hydrolytic stability was assessed at physiological pH. Then, the bioavailability was evaluated by orally administering the mPEG-DXM conjugate in rabbits and comparing to that obtained by the intravenous route. The area under the concentration-time curve (AUC) of PEGylated drug was twice as large as that of the parent drug after oral administration. In conclusion, PEGylated DXM was useful for maintaining the DXM concentration in the blood after oral administration.