Synthesis and controlled drug delivery studies of a novel Ubiquinol-Polyethylene glycol-Vitamin E adduct.

CoQ10 and Vitamin E are used in medicinal applications, but they are both lipophilic molecules and the poor solubility in aqueous media results in an inefficient administration, poor bioavailability and potential toxicity. A mixed conjugate Ubiquinol-Polyethylene glycol-Vitamin E was synthesized and characterized to improve the bioavailability of CoQ10 and Vitamin E. The synthesized mixed PEG conjugate was characterized by 1H NMR spectroscopy and MALDI spectrometry. The in vitro release of the conjugate was measured at various pH conditions and in human plasma and the evaluation of free CoQ10 and Vitamin E were also conducted. The obtained results demonstrated that more CoQ10 and Vitamin E were released from PEG conjugate at pH 7.4 and in plasma within the 24 h. The antioxidant activity evaluation was carried out by DPPH assay. Our results indicated that the chemical modification after esterification with PEG of the two drugs Ubiquinol and Vitamin E doesn't significantly affected their antioxidant potential.

Characterization of Arabica and Robusta volatile coffees composition by reverse carrier gas headspace gas chromatography-mass spectrometry based on a statistical approach.

Nineteen samples of Arabica and 14 of Robusta coming from various plantation were analysed by dynamic headspace capillary gas chromatography-mass spectrometry to characterize the volatile fraction of green and roasted samples and the relationships of the same species with geographical origin. As concerns green beans, Arabica species appear characterized by high content of n-hexanol, furfural and amylformate, while Robusta species by greater content of ethylpyrazine, dimethylsulfone and 2-heptanone. Four variables, 4-methyl-2,3-dihydrofuran, n-hexanol, limonene and nonanal, appear involved in the characterization of the geographical origin of the analysed samples. The volatile fraction of the roasted Arabica samples, appear characterized by high content of pyridine, diacetyl, propylformate, acetone and 2,3-pentanedione, while Robusta samples by high content of methylbutyrate, 2,3-dimethylpyrazine and 3-hexanone. Considering geographical origin of the analysed samples, four compounds appear involved, in particular 2-butanone, methylbutyrate, methanol and ethylformate. Very accurate (error rate lower than 5%) rules to classify samples as Arabica or Robusta according to their compounds profile were developed.

Polysaccharides from Pleurotus eryngii var. elaeoselini (Agaricomycetes), a New Potential Culinary-Medicinal Oyster Mushroom from Italy.

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)-ß-D-Glcp-(1→]2}n 6 ↑ 1 α-D-Glcp (2) PELPS-A2: [→6)-ß-D-Glcp-(1→6)-ß-D-Glcp-(1→6)-ß-D-Glcp-(1→]n 3 ↑ 1 ß-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.

Imidazole and 1,2,4-Triazole-based Derivatives Gifted with Antitubercular Activity: Cytotoxicity and Computational Assessment.

BACKGROUND: Mycobacterium Tuberculosis (Mtb) is the causative pathogen of Tuberculosis (TB) and outbreaks are more common among immunosuppressed persons infected with HIV. The current treatment regimens are lengthy and toxic, yet the therapy has remained unchanged for many decades, so there is a need to find new structures with selective mechanism of action. Moreover, the increased incidence of severe disseminated infections produced by undiagnosed Multidrug-resistant (MDR), worsen clinical treatment and contribute the spread of the disease. OBJECTIVE: The aim of our study was to evaluate the potential of imidazole and triazole moieties for antimycobacterial activity, by synthesizing some 1-(1-(aryl)-2-(2,6-dichlorophenyl)hydrazono)ethyl- 1H-imidazole and 1H-1,2,4-triazole derivatives 2a-l. METHODS: The title compounds were obtained via classical organic synthesis. The antimicrobial activity was evaluated using the method of microdilution and the cytotoxicity assay was performed by MTT method. RESULTS: The results indicated that the presence of both the imidazole ring and that of the 2,6- dichlorosubstituted phenyl moiety, is more relevant for inhibitory activity against Mtb than the triazole nucleus and the unsubstituted phenyl ring. Among the series, (E)-1-(2-(5-chlorothiophen-2-yl)-2-(2- (2,6-dichlorophenyl)hydrazono)ethyl)-1H-imidazole derivative 2f and (Z)-1-(2-([1,1'-biphenyl]-4-yl)- 2-(2-(2,6-dichlorophenyl)hydrazono)ethyl]-1H-imidazole derivatives 2e exhibited a promising antimycobacterial property and the latter also displayed a safe cytotoxic profile. CONCLUSION: The synthesized compounds were studied for their antitubercular activity. Among the series, the compounds 2e and 2f appeared to be the most promising agents and, according to the docking assessment, the compounds could be CYP51 inhibitors. These evidences could be useful for the future development of new antimycobacterial derivatives targeting CYP51 with more specificity for the mycobacterial cell enzyme.

Structural Characterization of Polysaccharides of a Productive Strain of the Culinary-Medicinal King Oyster Mushroom, Pleurotus eryngii (Agaricomycetes), from Italy.

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 single-quantum correlation spectroscopy, and heteronuclear multiple-bond correlation spectroscopy), the structures of the repeating unit of PEPS-A1 and PEPS-A2 were established as follows: (l)PEPS-Al (α-glucan): [→6)-α-D-Glcp-(1→6)-α-D-Glcp-(l→]n; and (2) PEPS-A2 (ß-glucan): [→6)-ß-D-Glcp-(1→6)-ß-D-Glcp-(l→]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 (HT-29). 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.

Improvement of Chemical and Physical Properties and Antioxidant Evaluation of Eugenol - PEG adduct.

Eugenol (EU) - PEG adduct was synthesized to improve the chemical and physical properties of eugenol. The phenolic group was covalently bound to the carboxyl group of PEG and the release kinetics were studied in vitro in buffer solution at pH 7.4, in simulated gastric fluid and in mouse plasma. Studies in vitro on the release of the parent drug from the prodrug in various media indicate that the adduct may be sufficiently stable to pass intact into the gastrointestinal tract and release EU into the circulation. The antioxidant activity of PEG-EU adduct was also evaluated. Scavenging activity was absent in the original PEG-EU adduct but gradually increased on the basis of drug delivery.

Antioxidant Properties of Oak Bracket Mushroom, Pseudoinonotus dryadeus (Higher Basidiomycetes): A Mycochemical Study.

A complex mixture of free fatty acids (1), cerevisterol (2), a sphingosine (3), and a complex mixture of diacylglycerophospholipids (4) were isolated from the fruiting body of the basidiomycete mushroom Pseudoinonotus dryadeus and subjected to spectroscopic analyses. The antioxidant activities of the whole extract of the fungus, of the isolated fractions, and of compounds 1-4 were evaluated in two in vitro model systems: 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and superoxide anion. In each systems, the extract of fungus and compound 2 showed the same free radical scavenging activity (with SC50 data of 18.27 µg/mL and 5.75 µg/mL, respectively) compared with the positive control quercetin (DPPH assay). Compounds 1-4 were isolated from P. dryadeus for the first time.

Lipid Metabolites from the Mushroom Meripilus giganteus.

The phytochemical investigation of the methanolic extract of the white rot fungus Meripilus giganteus resulted in the isolation and identification of complex mixtures of free fatty acids (1), monoacylglycerols (2), cerebrosides (3), ergosterol (4) and ergosterol peroxide (5). The structures of the isolated lipid metabolites (1-5) were determined by chemical and spectroscopic methods. The antioxidant activity of the whole MeOH extract of the fungus was evaluated through in vitro model systems, such as 2,2-diphenyl-l-picrylhydrazyl (DPPH) and superoxide anion. In all two systems, the results indicated that the extract of the fungus showed the same free-radical-scavenging activity with SC50 data of 47.70 µg/mL, compared with the positive control quercetin (DPPH assay). None of the isolated compounds (1-5) showed a significant activity. Compounds 2-4 were isolated from Meripilus giganteus for the first time.

PEG-Ursolic Acid Conjugate: Synthesis and In Vitro Release Studies.

A highly water-soluble macromolecular compound of ursolic acid with monomethoxypoly(ethylene glycol) (mPEG) was prepared. The physicochemical properties and stabilities under different conditions were investigated. By PEG conjugation, greatly increased water solubility was obtained, and the results showed that this conjugate was a potential prodrug for the oral delivery of ursolic acid.

Anticancer activity of cationic porphyrins in melanoma tumour-bearing mice and mechanistic in vitro studies.

BACKGROUND: Porphyrin TMPyP4 (P4) and its C14H28-alkyl derivative (C14) are G-quadruplex binders and singlet oxygen (1O2) generators. In contrast, TMPyP2 (P2) produces 1O2 but it is not a G-quadruplex binder. As their photosensitizing activity is currently undefined, we report in this study their efficacy against a melanoma skin tumour and describe an in vitro mechanistic study which gives insights into their anticancer activity. METHODS: Uptake and antiproliferative activity of photoactivated P2, P4 and C14 have been investigated in murine melanoma B78-H1 cells by FACS, clonogenic and migration assays. Apoptosis was investigated by PARP-1 cleavage and annexin-propidium iodide assays. Biodistribution and in vivo anticancer activity were tested in melanoma tumour-bearing mice. Porphyrin binding and photocleavage of G-rich mRNA regions were investigated by electrophoresis and RT-PCR. Porphyrin effect on ERK pathway was explored by Western blots. RESULTS: Thanks to its higher lipophylicity C14 was taken up by murine melanoma B78-H1 cells up to 30-fold more efficiently than P4. When photoactivated (7.2 J/cm2) in B78-H1 melanoma cells, P4 and C14, but not control P2, caused a strong inhibition of metabolic activity, clonogenic growth and cell migration. Biodistribution studies on melanoma tumour-bearing mice showed that P4 and C14 localize in the tumour. Upon irradiation (660 nm, 193 J/cm2), P4 and C14 retarded tumour growth and increased the median survival time of the treated mice by ~50% (P <0.01 by ANOVA), whereas porphyrin P2 did not. The light-dependent mechanism mediated by P4 and C14 is likely due to the binding to and photocleavage of G-rich quadruplex-forming sequences within the 5'-untranslated regions of the mitogenic ras genes. This causes a decrease of RAS protein and inhibition of downstream ERK pathway, which stimulates proliferation. Annexin V/propidium iodide and PARP-1 cleavage assays showed that the porphyrins arrested tumour growth by apoptosis and necrosis. C14 also showed an intrinsic light-independent anticancer activity, as recently reported for G4-RNA binders. CONCLUSIONS: Porphyrins P4 and C14 impair the clonogenic growth and migration of B78-H1 melanoma cells and inhibit melanoma tumour growth in vivo. Evidence is provided that C14 acts through light-dependent (mRNA photocleavage) and light-independent (translation inhibition) mechanisms.

Nitric oxide-mediated activity in anti-cancer photodynamic therapy.

Cell recurrence in cancer photodynamic therapy (PDT) is an important issue that is poorly understood. It is becoming clear that nitric oxide (NO) is a modulator of PDT. By acting on the NF-κB/Snail/RKIP survival/anti-apoptotic loop, NO can either stimulate or inhibit apoptosis. We found that pheophorbide a/PDT (Pba/PDT) induces the release of NO in B78-H1 murine amelanotic melanoma cells in a concentration-dependent manner. Low-dose PDT induces low NO levels by stimulating the anti-apoptotic nature of the above loop, whereas high-dose PDT stimulates high NO levels inhibiting the loop and activating apoptosis. When B78-H1 cells are treated with low-dose Pba/PDT and DETA/NO, an NO-donor, intracellular NO increases and cell growth is inhibited according to scratch-wound and clonogenic assays. Western blot analyses showed that the combined treatment reduces the expression of the anti-apoptotic NF-κB and Snail gene products and increases the expression of the pro-apoptotic RKIP gene product. The combined effect of Pba and DETA/NO was also tested in C57BL/6 mice bearing a syngeneic B78-H1 melanoma. We used pegylated Pba (mPEG-Pba) due to its better pharmacokinetics compared to free Pba. mPEG-Pba (30 mg/Kg) and DETA/NO (0.4 mg/Kg) were i.p. injected either as a single molecule or in combination. After photoactivation at 660 nM (fluence of 193 J/cm(2)), the combined treatment delays tumor growth more efficiently than each individual treatment (p<0.05). Taken together, our results showed that the efficacy of PDT is strengthened when the photosensitizer is used in combination with an NO donor.

Chemical composition and functional characterisation of commercial pumpkin seed oil.

BACKGROUND: Pumpkin (Cucurbita pepo L.) seed oil is a common product in Slovenia, Hungary and Austria and is considered a preventive agent for various pathologies, particularly prostate diseases. These properties are related to its high content of carotenoids and liposoluble vitamins. In this study the carotenoid (lutein and zeaxanthin), vitamin E (α- and γ-tocopherol) and fatty acid contents of 12 samples of commercial pumpkin seed oil were investigated together with the composition of the volatile fraction resulting from the roasting process. RESULTS: The aromatic profile obtained from the commercial samples was directly related to the intensity of the roasting process of the crushed pumpkin seeds. The roasting temperature played a crucial role in the concentrations of volatile substances originating from Strecker degradation, lipid peroxidation and Maillard reaction. CONCLUSION: The findings suggest that high-temperature roasting leads to the production of an oil with intense aromatic characteristics, while mild conditions, generally employed to obtain an oil with professed therapeutic characteristics, lead to a product with minor characteristic pumpkin seed oil aroma. The nutraceutical properties of the product are confirmed by the high content of α- and γ-tocopherol and carotenoids.

The PDT activity of free and pegylated pheophorbide a against an amelanotic melanoma transplanted in C57/BL6 mice.

Pheophorbide a (Pba) is a chlorophyll catabolite that has been proposed as photosensitizer in photodynamic therapy. In a previous study we conjugated Pba to monomethoxy-polyethylene glycol (mPEG-Pba), to increase its solubility and pharmacokinetics. Here, we compare the photodynamic therapy efficacy of free Pba and mPEG-Pba to cure a subcutaneous amelanotic melanoma transplanted in C57/BL6 mice. The photosensitizers, i.p. injected (30 mg/kg), showed no toxicity when the animals were kept in the dark. But, after photoactivation with a 660 nm laser (fluence of 193 J/cm(2)), both photosensitizers, in particular mPEG-Pba, showed a strong efficacy to cure the tumor, both in terms of tumor growth delay and increase of Kaplan-Meier median survival time. Together, our in vivo data demonstrate that mPEG-conjugated Pba is a promising photosensitizer for the photodynamic therapy of cancer.

Pharmacokinetic analysis of multi PEG-theophylline conjugates.

In the attempt of prolonging the effect of drugs, a new branched, high-molecular weight multimeric poly(ethylene glycol) (MultiPEG), synthesized with a simple assembling procedure that devised the introduction of functional groups with divergent and selective reactivity, was employed as drug carrier. In particular, the attention was focused on the study of theophylline (THEO) and THEO-MultiPEG conjugates pharmacokinetic after oral administration in rabbit. Pharmacokinetic behavior was studied according to an ad hoc developed mathematical model accounting for THEO-MultiPEG in vivo absorption and decomposition into drug (THEO) and carrier (MultiPEG). The branched high-molecular weight MultiPEG proved to be a reliable drug delivery system able to prolong theophylline staying in the blood after oral administration of a THEO-MultiPEG solution. The analysis of experimental data by means of the developed mathematical model revealed that the prolongation of THEO effect was essentially due to the low THEO-MultiPEG permeability in comparison to that of pure THEO.

Bioactive lipids metabolites in Amanita virosa.

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%), but showed no inhibitory activity on trypsin. On the basis of inhibition selectivity between thrombin and trypsin and potency of thrombin inhibition, A. virosa constitutes a good starting material for the isolation of further compounds that are active against thrombin. Bioassay oriented fractionation of the extract of A. virosa led to the isolation of a complex mixture of triglycerides (TGs), monoacylglycerols (MAGs), free fatty acids (FAs) and ergosterol. The structures of the isolated lipids metabolites were determined on the basis of chemical and spectroscopic evidences.

Melt extruded helical waxy matrices as a new sustained drug delivery system.

The aim of this research was to prepare helical and cylindrical extrudates by melt extrusion and to evaluate their potential as sustained release dosage form. The systems contained theophylline as water-soluble model drug and microcrystalline wax as thermoplastic binder. The temperature suitable to ensure a successful extrusion process of formulations containing the wax in three different percentages was found to be below the melting point of the excipient. After the production of the extrudates in three different helical shapes (having 2, 3 and 4 blades) and a classical cylindrical shape, the systems were studied by means of X-ray powder diffraction and differential scanning calorimetry to check possible variations of the solid state of the drug during the thermal process. The morphology and chemical composition of the surface of the extrudates were examined by Scanning Electron Microscopy/Energy Dispersive X-ray Microanalysis to evaluate the presence of the drug on the surface of the extrudates and to monitor changes on the aspect of the waxy matrix during dissolution. Then, the different systems were analysed from the in vitro dissolution point of view to study the influence of the shape and of the composition on the drug release. An in vivo pilot study on the best performing system (helix with 3 blades) was carried out on five healthy volunteers and monitoring the intestinal transit by X-ray images. The resulting plasma profiles were analysed by means of a suitable pharmacokinetic analysis. Finally, an ad hoc mathematical model was developed to perform an accurate description of the in vitro release and in vivo performance of the 3-blades helical system.

Conjugated PDT drug: photosensitizing activity and tissue distribution of PEGylated pheophorbide a.

The design of new photosensitizers with enhanced phototoxicity and pharmacokinetic properties remains a central challenge for cancer photodynamic therapy (PDT). In this study, Pheophorbide a (Pba) has been pegylated to methoxypolyethylene glycol (mPE G-Pba) to produce a soluble photosensitizer that exhibits a higher tissue distribution than free Pba. In vitro studies have shown that mPE G-Pba promotes a fairly strong photosensitizing effect in cancer cells, as previously observed for the unpegylated molecule. mPE G-Pba targets the mitochondria where, following photoactivation, ROS are produced which cause a cellular injury by lipid peroxidation. The effect of pegylation on the photosensitizer biodistribution has been examined in different selected organs of female mice, at different time points after intraperitoneal administration of the drug (50 µmol/Kg body weight). Other than free Pba, which showed a low tissue accumulation, mPE G-Pba has been detected in significant amounts (8 to 16 µg/ml) in liver, spleen, duodenum and kidney and, 3-5 hours after intraperitoneal injection, in moderate amounts (3 to 8 µg/ml) in brain and lung. In vivo optical imaging 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. Our results indicate that although pegylation somewhat decreases the phototoxicity, it significantly increases the drug solubility and tissue distribution and tumor uptake of mPE G-Pba, making the conjugate an interesting photosensitizer for PDT.

Cerebrosides with antiproliferative activity from Euphorbia peplis L.

Two new cerebrosides have been isolated from the whole plants of Euphorbia peplis L. The structures were established by FT-IR spectroscopy, FAB MS, EI-MS, ESI-MS, 1D and 2D NMR spectroscopy. The structures of the cerebrosides were characterized as 1-O-beta-d-glycosides of phytosphingosines, which comprised a common long-chain base, (2S, 3S, 4R, 8Z)-2-amino-8 (Z)-octadecene-1,3,4-triol with 2-hydroxy fatty acids of varying chain lengths (C25, C22) linked to the amino group. The isolated compounds were shown to possess significant antiproliferative properties against cultured human tumor cell lines KB and IMR-32.

Synthesis of 4-thiophen-2'-yl-1,4-dihydropyridines as potentiators of the CFTR chloride channel.

The gating of the CFTR chloride channel is altered by a group of mutations that cause cystic fibrosis. This gating defect may be corrected by small molecules called potentiators. Some 1,4-dihydropyridine (DHP) derivatives, bearing a thiophen-2-yl and a furanyl ring at the 4-position of the nucleus, were prepared and tested as CFTR potentiators. In particular, we evaluated the ability of novel DHPs to enhance the activity of the rescued DeltaF508-CFTR as measured with a functional assay based on the halide-sensitive yellow fluorescent protein. Most DHPs showed an effect comparable to or better than that of the reference compound genistein. The potency was instead significantly improved, with some compounds, such as 3g, 3h, 3n, 4a, 4b, and 4d, having a half effective concentration in the submicromolar range. CoMFA analysis gave helpful suggestions to improve the activity of DHPs.

In vitro behavior of multifunctionalized fullerene-warfarin conjugates.

In this study we have covalently linked the anticoagulant warfarin to polyfunctionalized fullerenes. The objective was to explore the possibility of modifying the biological profile of a drug by covalent binding to functionalized fullerene. We have chosen warfarin as a model compound because it a widely used drug. We have analyzed the stability in vitro of the conjugates and found that the drug is released from the carbon support only after incubation in mouse plasma.