Evaluation of the Antifungal and Antiproliferative Properties of the Lichen Roccella tinctoria DC. Extracts and Main Components.

In this work, phytochemical analysis on different extracts of Roccella tinctoria DC. was reported using different techniques with respect to the past. Twenty volatile and three non-volatile compounds were identified, some of which were found in this species for the first time. The methanolic extracts and their non-volatile components were then evaluated for their antitumor effects in cancerous A549 and Mz-ChA-1 cells and for their tolerability in non-cancerous BEAS-2B and H69 cells, showing IC50 values from 94.6 µg/mL to 416.4 µg/mL, in general. The same extracts and compounds were also tested for their antifungal effects in Candida albicans, with only compound 2 being active, with an MIC50 value of 87 µg/mL. In addition, they were tested for their anti-Candida adhesion activity, anti-Candida biofilm formation, and anti-Candida mature biofilm inhibition, with efficacy percentages generally above 50% but not for all of them. Lastly, the DF3 extract and compounds 1-2 were tested in vivo according to the Galleria mellonella survival assay, showing positive mortality rates above 50% at different concentrations. All these biological assays were conducted on this species for the first time. Comparisons with other lichens and compounds were also presented and discussed.

Phytochemical Analysis and In Vitro Antileukemic Activity of Alkaloid-Enriched Extracts from Vinca sardoa (Stearn) Pignatti.

Vinca sardoa (Stearn) Pignatti, known as Sardinian periwinkle, is widely diffused in Sardinia (Italy). This species contains indole alkaloids, which are known to have a great variety of biological activities. This study investigated the antileukemic activity against a B lymphoblast cell line (SUP-B15) of V. sardoa alkaloid-rich extracts obtained from plants grown in Italy, in Iglesias (Sardinia) and Rome (Latium). All the extracts showed a good capacity to induce reductions in cell proliferation of up to 50% at the tested concentrations (1-15 µg/mL). Moreover, none of the extracts showed cytotoxicity on normal cells at all the studied concentrations.

Phytochemicals as Immunomodulatory Agents in Melanoma.

Cutaneous melanoma is an immunogenic highly heterogenic tumor characterized by poor outcomes when it is diagnosed late. Therefore, immunotherapy in combination with other anti-proliferative approaches is among the most effective weapons to control its growth and metastatic dissemination. Recently, a large amount of published reports indicate the interest of researchers and clinicians about plant secondary metabolites as potentially useful therapeutic tools due to their lower presence of side effects coupled with their high potency and efficacy. Published evidence was reported in most cases through in vitro studies but also, with a growing body of evidence, through in vivo investigations. Our aim was, therefore, to review the published studies focused on the most interesting phytochemicals whose immunomodulatory activities and/or mechanisms of actions were demonstrated and applied to melanoma models.

Recent Advances in Recovery of Lycopene from Tomato Waste: A Potent Antioxidant with Endless Benefits.

Growing attention to environmental protection leads food industries to adopt a model of "circular economy" applying safe and sustainable technologies to recover, recycle and valorize by-products. Therefore, by-products become raw material for other industries. Tomato processing industry produces significant amounts of by-products, consisting of skins and seeds. Tomato skin is very rich in lycopene, and from its seeds, high nutritional oil can be extracted. Alternative use of the two fractions not only could cut disposal costs but also allow one to extract bioactive compounds and an oil with a high nutritional value. This review focused on the recent advance in extraction of lycopene, whose beneficial effects on health are widely recognized.

Anti-Tumoral Effects of a (1H-Pyrrol-1-yl)Methyl-1H-Benzoimidazole Carbamate Ester Derivative on Head and Neck Squamous Carcinoma Cell Lines.

Nocodazole is an antineoplastic agent that exerts its effects by depolymerizing microtubules. Herein we report a structural analog of nocodazole, a (1H-pyrrol-1-yl)methyl-1H-benzoimidazole carbamate ester derivative, named RDS 60. We evaluated the antineoplastic properties of RDS 60 in two human head and neck squamous cell carcinoma (HNSCC) cell lines and we found that this compound significantly inhibited replication of both HNSCC cell lines without inducing any important cytotoxic effect on human dermal fibroblasts and human keratinocytes. The treatment of HNSCC cell lines with 1 µM RDS 60 for 24 h stopped development of normal bipolar mitotic spindles and, at the same time, blocked the cell cycle in G2/M phase together with cytoplasmic accumulation of cyclin B1. Consequently, treatment with 2 µM RDS 60 for 24 h induced the activation of apoptosis in both HNSCC cell lines. Additionally, RDS 60 was able to reverse the epithelial-mesenchymal transition and to inhibit cell migration and extracellular matrix infiltration of both HNSCC cell lines. The reported results demonstrate that this compound has a potent effect in blocking cell cycle, inducing apoptosis and inhibiting cell motility and stromal invasion of HNSCC cell lines. Therefore, the ability of RDS 60 to attenuate the malignancy of tumor cells suggests its potential role as an interesting and powerful tool for new approaches in treating HNSCC.

Design, synthesis and biological evaluation of a series of iron and copper chelating deferiprone derivatives as new agents active against Candida albicans.

Candida albicans, in specific conditions, is responsible of severe invasive systemic candidiasis that are related to its ability to produce biofilm on biological and artificial surfaces. Many studies reported the role of iron in fungal growth and virulence and the ability of metal chelating agents to interfere with C. albicans metabolism, virulence and biofilm formation. Here we report the activity of 3-hydroxy-1,2-dimethyl-4(1H)-pyridinone (deferiprone) derivatives against C. albicans planktonic cells and biofilm. Some of the studied compounds (2b and 3b) were able to chelate Fe(III) and Cu(II), and showed an interesting activity on planktonic cells (MIC50 of 32 µg/mL and 16 µg/mL respectively) and on biofilm formation (BMIC50 of 32 µg/mL and 16 µg/mL respectively) in cultured ATCC 10,231C. albicans; this activity was reduced, in a concentration dependent way, by the addition of Fe(III) and Cu(II) to the culture media. Furthermore, the most active compound 3b showed a low toxicity on Galleria mellonella larvae.

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.

Investigation of Commiphora myrrha (Nees) Engl. Oil and Its Main Components for Antiviral Activity.

The resinous exudate produced by Commiphora myrrha (Nees) Engl. is commonly known as true myrrh and has been used since antiquity for several medicinal applications. Hundreds of metabolites have been identified in the volatile component of myrrh so far, mainly sesquiterpenes. Although several efforts have been devoted to identifying these sesquiterpenes, the phytochemical analyses have been performed by gas-chromatography/mass spectrometry (GC-MS) where the high temperature employed can promote degradation of the components. In this work, we report the extraction of C. myrrha by supercritical CO2, an extraction method known for the mild extraction conditions that allow avoiding undesired chemical reactions during the process. In addition, the analyses of myrrh oil and of its metabolites were performed by HPLC and GC-MS. Moreover, we evaluated the antiviral activity against influenza A virus of the myrrh extracts, that was possible to appreciate after the addition of vitamin E acetate (α-tocopheryl acetate) to the extract. Further, the single main bioactive components of the oil of C. myrrha commercially available were tested. Interestingly, we found that both furanodienone and curzerene affect viral replication by acting on different steps of the virus life cycle.

Design, Synthesis and Biological Evaluation of New Pyrimidine Derivatives as Anticancer Agents.

BACKGROUND: Anticancer drug resistance is a challenging phenomenon of growing concern which arises from alteration in drug targets. Despite the fast speed of new chemotherapeutic agent design, the increasing prevalence of this phenomenon requires further research and treatment development. Recently, we reported a new aminopyrimidine compound-namely RDS 344-as a potential innovative anticancer agent. METHODS: Herein, we report the design, synthesis, and anti-proliferative activity of new aminopyrimidine derivatives structurally related to RDS 3442 obtained by carrying out substitutions at position 6 of the pyrimidine core and/or on the 2-aniline ring of our hit. The ability to inhibit cell proliferation was evaluated on different types of tumors, glioblastoma, triple-negative breast cancer, oral squamous cell carcinomas and colon cancer plus on human dermal fibroblasts chosen as control of normal cells. RESULTS: The most interesting compound was the N-benzyl counterpart of RDS 3442, namely 2a, that induced a significant decrease in cell viability in all the tested tumor cell lines, with EC50s ranging from 4 and 8 µM, 4-13 times more active of hit. CONCLUSIONS: These data suggest a potential role for this class of molecules as promising tool for new approaches in treating cancers of different histotype.

Towards a new application of amaranth seed oil as an agent against Candida albicans.

Amaranthus spp. (Amaranthaceae family), known as amaranth, are plants native of Central America, today produced in many parts of the world. due to their popularity popular as a health food. Because of its composition, amaranth can be considered to be attractive not only as a food but also for pharmaceutical and cosmetics uses. To date, antifungal activity of amaranth extracts has not been totally investigated, therefore the scope of this study was to evaluate the antifungal effect of the apolar fraction from Amaranthus cruentus L. seeds extract, alone and in association with antifungal drugs terbinafine, a common antifungal agent, which itself has only fungistatic effect on Candida albicans strains without exerting fungicidal activity. Our results demonstrate that this amaranth oil in combination with terbinafine has synergic fungistatic and fungicidal activity, with FICI of 0.466 and 0.496, respectively. No fungistatic and fungicidal activity of terbinafine alone at concentrations up to 64 µg/mL and amaranth oil alone at concentrations up to 2000 µg/mL, against all tested C. albicans strains, were observed. does not show activity towards Candida albicans strains but it can effectively potentiate the antifungal activity of terbinafine, a common antifungal agent which itself This result suggests the possible application of amaranth oil in the preparation of formulations with terbinafine for topical use.

Molecular design aided by random forests and synthesis of potent trypanocidal agents as cruzain inhibitors for Chagas disease treatment.

Cruzain is an established target for the identification of novel trypanocidal agents, but how good are in vitro/in vivo correlations? This work describes the development of a random forests model for the prediction of the bioavailability of cruzain inhibitors that are Trypanosoma cruzi killers. Some common properties that characterize drug-likeness are poorly represented in many established cruzain inhibitors. This correlates with the evidence that many high-affinity cruzain inhibitors are not trypanocidal agents against T. cruzi. On the other hand, T. cruzi killers that present typical drug-like characteristics are likely to show better trypanocidal action than those without such features. The random forests model was not outperformed by other machine learning methods (such as artificial neural networks and support vector machines), and it was validated with the synthesis of two new trypanocidal agents. Specifically, we report a new lead compound, Neq0565, which was tested on T. cruzi Tulahuen (ß-galactosidase) with a pEC50 of 4.9. It is inactive in the host cell line showing a selectivity index (SI = EC50cyto /EC50T. cruzi ) higher than 50.

Crystal structure of Leishmania mexicana cysteine protease B in complex with a high-affinity azadipeptide nitrile inhibitor.

Leishmania mexicana is an obligate intracellular protozoan parasite that causes the cutaneous form of leishmaniasis affecting South America and Mexico. The cysteine protease LmCPB is essential for the virulence of the parasite and therefore, it is an appealing target for antiparasitic therapy. A library of nitrile-based cysteine protease inhibitors was screened against LmCPB to develop a treatment of cutaneous leishmaniasis. Several compounds are sufficiently high-affinity LmCPB inhibitors to serve both as starting points for drug discovery projects and as probes for target validation. A 1.4 Å X ray crystal structure, the first to be reported for LmCPB, was determined for the complex of this enzyme covalently bound to an azadipeptide nitrile ligand. Mapping the structure-activity relationships for LmCPB inhibition revealed superadditive effects for two pairs of structural transformations. Therefore, this work advances our understanding of azadipeptidyl and dipeptidyl nitrile structure-activity relationships for LmCPB structure-based inhibitor design. We also tested the same series of inhibitors on related cysteine proteases cathepsin L and Trypanosoma cruzi cruzain. The modulation of these mammalian and protozoan proteases represents a new framework for targeting papain-like cysteine proteases.

N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design, synthesis and biochemical characterization.

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (Ki = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4'-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (Ki = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S.

Synthesis and matched molecular pair analysis of covalent reversible inhibitors of the cysteine protease CPB.

Cysteine protease B (CPB) can be targeted by reversible covalent inhibitors that could serve as antileishmanial compounds. Here, sixteen dipeptidyl nitrile derivatives were synthesized, tested against CPB, and analyzed using matched molecular pairs to determine the effects of stereochemistry and p-phenyl substitution on enzyme inhibition. The compound (S)-2-(((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)amino)-N-(1-cyanocyclopropyl)-3-phenylpropanamide (5) was the most potent CPB inhibitor (pKi = 6.82), which was also selective for human cathepsin B (pKi < 5). The inversion of the stereochemistry from S to R was more detrimental to potency when placed at the P2 position than at P3. The p-Br derivatives were more potent than the p-CH3 and p-OCH3 derivatives, probably due to intermolecular interactions with the S3 subsite.

New deferiprone derivatives as multi-functional cholinesterase inhibitors: design, synthesis and in vitro evaluation.

In order to obtain multi-functional molecules for Alzheimer's disease, a series of deferiprone derivatives has been synthesized and evaluated in vitro with the hypothesis that they can restore the cholinergic tone and attenuate the dyshomeostasis of the metals mainly involved in the pathology. These compounds were designed as dual binding site AChE inhibitors: they possess an arylalkylamine moiety connected via an alkyl chain to a 3-hydroxy-4-pyridone fragment, to allow the simultaneous interaction with catalytic active site (CAS) and peripheral anionic site (PAS) of the enzyme. Deferiprone moiety and 2-aminopyridine, 2-aminopyrimidine or 2,4-diaminopyrimidine groups have been incorporated into these compounds, in order to obtain molecules potentially able to chelate bio-metals colocalized in Aß plaques and involved in the generation of radical species. Synthesized compounds were tested by enzymatic inhibition studies towards EeAChE and eqBChE using Ellman's method. The most potent EeAChE inhibitor is compound 5a, with a Ki of 788 ± 51 nM, while the most potent eqBChE inhibitors are compounds 12 and 19, with Ki values of 182 ± 18 nM and 258 ± 25 nM respectively. Selected compounds, among the most potent cholinesterases inhibitors, were able to form complex with iron and in some cases with copper and zinc. Moreover, these compounds were characterized by low toxicity on U-87 MG Cell Line from human brain (glioblastoma astrocytoma).

Assessment of the Cruzain Cysteine Protease Reversible and Irreversible Covalent Inhibition Mechanism.

Reversible and irreversible covalent ligands are advanced cysteine protease inhibitors in the drug development pipeline. K777 is an irreversible inhibitor of cruzain, a necessary enzyme for the survival of the Trypanosoma cruzi (T. cruzi) parasite, the causative agent of Chagas disease. Despite their importance, irreversible covalent inhibitors are still often avoided due to the risk of adverse effects. Herein, we replaced the K777 vinyl sulfone group with a nitrile moiety to obtain a reversible covalent inhibitor (Neq0682) of cysteine protease. Then, we used advanced experimental and computational techniques to explore details of the inhibition mechanism of cruzain by reversible and irreversible inhibitors. The isothermal titration calorimetry (ITC) analysis shows that inhibition of cruzain by an irreversible inhibitor is thermodynamically more favorable than by a reversible one. The hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) and Molecular Dynamics (MD) simulations were used to explore the mechanism of the reaction inhibition of cruzain by K777 and Neq0682. The calculated free energy profiles show that the Cys25 nucleophilic attack and His162 proton transfer occur in a single step for a reversible inhibitor and two steps for an irreversible covalent inhibitor. The hybrid QM/MM calculated free energies for the inhibition reaction correspond to -26.7 and -5.9 kcal mol-1 for K777 and Neq0682 at the MP2/MM level, respectively. These results indicate that the ΔG of the reaction is very negative for the process involving K777, consequently, the covalent adduct cannot revert to a noncovalent protein-ligand complex, and its binding tends to be irreversible. Overall, the present study provides insights into a covalent inhibition mechanism of cysteine proteases.

On the intrinsic reactivity of highly potent trypanocidal cruzain inhibitors.

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Hence, peptidomimetic cruzipain inhibitors having a reactive group (known as warhead) are subject to continuous studies to discover novel antichagasic compounds. Here, we evaluated how different warheads for a set of structurally similar related compounds could inhibit the activity of cruzipain and, ultimately, their trypanocidal effect. We first investigated in silico the intrinsic reactivity of these compounds by applying the Fukui index to correlate it with the enzymatic affinity. Then, we evaluated their potency against T. cruzi (Y and Tulahuen strains), which revealed the reversible cruzain inhibitor Neq0656 as a better trypanocidal agent (ECY.strain 50 = 0.1 µM; SI = 58.4) than the current drug benznidazole (ECY.strain 50 = 5.1 µM; SI > 19.6). We also measured the half-life time by HPLC analysis of three lead compounds in the presence of glutathione and cysteine to experimentally assess their intrinsic reactivity. Results clearly illustrated the reactivity trend for the warheads (azanitrile > aldehyde > nitrile), where the aldehyde displayed an intermediate intrinsic reactivity. Therefore, the aldehyde bearing peptidomimetic compounds should be subject for in-depth evaluation in the drug discovery process.

Comparison of different methods for the extraction of cannabinoids from cannabis.

Cannabis oils, namely concentrated cannabis extracts, are getting plenty of attention because of their therapeutic potential for treatment of patients with cancer, HIV, multiple sclerosis and several other pathologies. Here we propose the use of ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) as alternative methods to the current protocols followed by pharmacists, the only authorized to manipulate standardized Cannabis. A third method, consisting of the use of Tween 20 as surfactant, was considered. Our best extraction methodology for commercial hemp extraction was applied to medicinal cannabis. Here we report the results obtained for 'Eletta campana', 'Carmagnola selezionata', Bediol®, FM2® and Bedrocan®.

Anti-Inflammatory and Antioxidant Properties of Dehydrated Potato-Derived Bioactive Compounds in Intestinal Cells.

Inflammation and oxidative stress are always more recognized as responsible for chronic disease at the intestinal level. Currently, a growing interest is addressed to the discovery of diet-derived products which have anti-inflammatory and antioxidant properties. This work aims to characterize the pharmacological potential of dehydrated potatoes. For this purpose, a simulated gastrointestinal digestion was carried out. The bioaccessible peptides were fractionated on the basis of their molecular weight and tested on intestinal epithelial cells (IEC-6) under oxidative and inflammatory conditions. Our results demonstrate that the tested peptide fractions were able to significantly inhibit tumor necrosis factor-α release and cycloxygenase-2 and inducible nitric oxide synthase expression. The tested peptides also showed significant antioxidant activity, being able to both reduce reactive oxygen species (ROS) release, also from mitochondria, and nitrotyrosine formation, and increase the antioxidant response by heme oxygenase-1 and superoxide dismutase expression. Moreover, the peptide fractions were able to significantly increase the wound repair in IEC-6. The obtained results indicate the anti-inflammatory and antioxidant potential of dehydrated potatoes at the intestinal level.

Synthesis and structure-activity relationship of nitrile-based cruzain inhibitors incorporating a trifluoroethylamine-based P2 amide replacement.

The structure-activity relationship for nitrile-based cruzain inhibitors incorporating a P2 amide replacement based on trifluoroethylamine was explored by deconstruction of a published series of inhibitors. It was demonstrated that the P3 biphenyl substituent present in the published inhibitor structures could be truncated to phenyl with only a small loss of affinity. The effects of inverting the configuration of the P2 amide replacement and linking a benzyl substituent at P1 were observed to be strongly nonadditive. We show that plotting affinity against molecular size provides a means to visualize both the molecular size efficiency of structural transformations and the nonadditivity in the structure-activity relationship. We also show how the relationship between affinity and lipophilicity, measured by high-performance liquid chromatography with an immobilized artificial membrane stationary phase, may be used to normalize affinity with respect to lipophilicity.