Repositioning of Small Molecules through the Inverse Virtual Screening in silico Tool: Case of Benzothiazole-Based Inhibitors of Soluble Epoxide Hydrolase (sEH).

Computational techniques accelerate drug discovery by identifying bioactive compounds for specific targets, optimizing molecules with moderate activity, or facilitating the repositioning of inactive items onto new targets. Among them, the Inverse Virtual Screening (IVS) approach is aimed at the evaluation of one or a small set of molecules against a panel of targets for addressing target identification. In this work, a focused library of benzothiazole-based compounds was re-investigated by IVS. Four items, originally synthesized and tested on bromodomain-containing protein 9 (BRD9) but yielding poor results, were critically re-analyzed, disclosing only a partial fit with 3D structure-based pharmacophore models, which, in the meanwhile, were developed for this target. Afterwards, these compounds were re-evaluated through IVS on a panel of proteins involved in inflammation and cancer, identifying soluble epoxide hydrolase (sEH) as a putative interacting target. Three items were subsequently confirmed as able to inhibit sEH activity, spanning from 70% up to 30% when tested at 10 µM. Finally, one benzothiazole-based compound emerged as the most promising inhibitor featuring an IC50 in the low micromolar range (IC50 = 6.62 ± 0.13 µM). Our data confirm IVS as a predictive tool for accelerating the target identification and repositioning processes.

NMR Metabolomics and Chemometrics of Commercial Varieties of Phaseolus vulgaris L. Seeds from Italy and In Vitro Antioxidant and Antifungal Activity.

The metabolite fingerprinting of four Italian commercial bean seed cultivars, i.e., Phaseolus Cannellino (PCANN), Controne (PCON), Vellutina (PVEL), and Occhio Nero (PON), were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate data analysis. The hydroalcoholic and organic extract analysis disclosed more than 32 metabolites from various classes, i.e., carbohydrates, amino acids, organic acids, nucleosides, alkaloids, and fatty acids. PVEL, PCON, and PCANN varieties displayed similar chemical profiles, albeit with somewhat different quantitative results. The PON metabolite composition was slightly different from the others; it lacked GABA and pipecolic acid, featured a higher percentage of malic acid than the other samples, and showed quantitative variations of several metabolites. The lipophilic extracts from all four cultivars demonstrated the presence of omega-3 and omega-6 unsaturated fatty acids. After the determination of the total phenolic, flavonoids, and condensed tannins content, in vitro antioxidant activity was then assessed using the DPPH scavenging activity, the ABTS scavenging assay, and ferric-reducing antioxidant power (FRAP). Compared to non-dark seeds (PCON, PCANN), brown seeds (PVEL, PON) featured a higher antioxidant capacity. Lastly, only PON extract showed in vitro antifungal activity against the sclerotia growth of S. rolfsii, by inhibiting halo growth by 75%.

Identification of 2-Aminoacyl-1,3,4-thiadiazoles as Prostaglandin E2 and Leukotriene Biosynthesis Inhibitors.

The application of a multi-step scientific workflow revealed an unprecedented class of PGE2/leukotriene biosynthesis inhibitors with in vivo activity. Specifically, starting from a combinatorial virtual library of ∼4.2 × 105 molecules, a small set of compounds was identified for the synthesis. Among these, four novel 2-aminoacyl-1,3,4-thiadiazole derivatives (3, 6, 7, and 9) displayed marked anti-inflammatory properties in vitro by strongly inhibiting PGE2 biosynthesis, with IC50 values in the nanomolar range. The hit compounds also efficiently interfered with leukotriene biosynthesis in cell-based systems and modulated IL-6 and PGE2 biosynthesis in a lipopolysaccharide-stimulated J774A.1 macrophage cell line. The most promising compound 3 showed prominent in vivo anti-inflammatory activity in a mouse model, with efficacy comparable to that of dexamethasone, attenuating zymosan-induced leukocyte migration in mouse peritoneum with considerable modulation of the levels of typical pro-/anti-inflammatory cytokines.

Chemical Profiles, In Vitro Antioxidant and Antifungal Activity of Four Different Lavandula angustifolia L. EOs.

Lavandula angustifolia L., known as lavender, is an economically important Lamiaceae due to the production of essential oils (EOs) for the food, cosmetic, pharmaceutical and medical industries. The purpose of this study was to determine the chemical composition of EOs isolated from four inflorescences of L. angustifolia L. collected in different geographical areas: central-southern Italy (LaCC, LaPE, LaPS) and southern France (LaPRV). The essential oils, obtained by steam distillation from plants at the full flowering stage, were analyzed using gas chromatography coupled with mass spectrometry (GC-MS). More than 70 components identified in each sample showed significant variability among the main constituents. The four EOs analyzed contained the following as main component: linalool (from 30.02% to 39.73%), borneol (13.65% in LaPE and 16.83% in La PS), linalyl acetate (24.34% in LaCC and 31.07% in LaPRV). The EOs were also evaluated for their in vitro antifungal activity against two white rot fungi (Phanerochaete chrysosporium and Trametes cingulata) as potential natural biodeteriogens in the artworks field, and against Sclerotium rolfsii, Botrytis cinerea and Fusarium verticilloides responsible for significant crop yield losses in tropical and subtropical areas. The results confirm a concentration-dependent toxicity pattern, where the fungal species show different sensitivity to the four EOs. The in vitro antioxidant activity by DPPH assay showed better scavenging activity on LaCC (IC50 26.26 mg/mL) and LaPRV (IC50 33.53 mg/mL), followed by LaPE (IC50 48.00 mg/mL) and LaPS (IC50 49.63 mg/mL). The potential application of EOs as a green method to control biodeterioration phenomena on a work of art on wood timber dated 1876 was evaluated.

Phytochemical Analysis of the Methanolic Extract and Essential Oil from Leaves of Industrial Hemp Futura 75 Cultivar: Isolation of a New Cannabinoid Derivative and Biological Profile Using Computational Approaches.

Cannabis sativa L. is a plant belonging to the Cannabaceae family, cultivated for its psychoactive cannabinoid (Δ9-THC) concentration or for its fiber and nutrient content in industrial use. Industrial hemp shows a low Δ9-THC level and is a valuable source of phytochemicals, mainly represented by cannabinoids, flavones, terpenes, and alkaloids, with health-promoting effects. In the present study, we investigated the phytochemical composition of leaves of the industrial hemp cultivar Futura 75, a monoecious cultivar commercially used for food preparations or cosmetic purposes. Leaves are generally discarded, and represent waste products. We analyzed the methanol extract of Futura 75 leaves by HPLC and NMR spectroscopy and the essential oil by GC-MS. In addition, in order to compare the chemical constituents, we prepared the water infusion. One new cannabinoid derivative (1) and seven known components, namely, cannabidiol (2), cannabidiolic acid (3), ß-cannabispirol (4), ß-cannabispirol (5), canniprene (6), cannabiripsol (7), and cannflavin B (8) were identified. The content of CBD was highest in all preparations. In addition, we present the outcomes of a computational study focused on elucidating the role of 2α-hydroxy-Δ3,7-cannabitriol (1), CBD (2), and CBDA (3) in inflammation and thrombogenesis.

In Silico, In Vitro, and In Vivo Analysis of Tanshinone IIA and Cryptotanshinone from Salvia miltiorrhiza as Modulators of Cyclooxygenase-2/mPGES-1/Endothelial Prostaglandin EP3 Pathway.

Tanshinone IIA (TIIA) and cryptotanshinone (CRY) from Salvia miltiorrhiza Bunge were investigated for their inhibitory activity against the cyclooxygenase-2 (COX-2)/microsomal prostaglandin E synthase-1 (mPGES-1)/endothelial prostaglandin 3 (EP3) pathway using in silico, in vitro, in vivo, and ex vivo assays. From the analysis of the docking poses, both diterpenoids were able to interact significantly with COX-2, 5-lipoxygenase (5-LO), platelet-activating factor receptor (PAFR), and mPGES-1. This evidence was further corroborated by data obtained from a cell-free assay, where CRY displayed a significant inhibitory potency against mPGES-1 (IC50 = 1.9 ± 0.4 µM) and 5-LO (IC50 = 7.1 µM), while TIIA showed no relevant inhibition of these targets. This was consistent with their activity to increase mice bleeding time (CRY: 2.44 ± 0.13 min, p ≤ 0.001; TIIA: 2.07 ± 0.17 min p ≤ 0.01) and with the capability to modulate mouse clot retraction (CRY: 0.048 ± 0.011 g, p ≤ 0.01; TIIA: 0.068 ± 0.009 g, p ≤ 0.05). For the first time, our results show that TIIA and, in particular, CRY are able to interact significantly with the key proteins involved not only in the onset of inflammation but also in platelet activity (and hyper-reactivity). Future preclinical and clinical investigations, together with this evidence, could provide the scientific basis to consider these compounds as an alternative therapeutic approach for thrombotic- and thromboembolic-based diseases.

Biological Profile of Two Gentiana lutea L. Metabolites Using Computational Approaches and In Vitro Tests.

Natural products have been the main source of bioactive molecules for centuries. We tested the biological profile of two metabolites extracted from Gentiana lutea L. by means of computational techniques and in vitro assays. The two molecules (loganic acid and gentiopicroside) were tested in silico using an innovative technique, named Inverse Virtual Screening (IVS), to highlight putative partners among a panel of proteins involved in inflammation and cancer events. A positive binding with cyclooxygenase-2 (COX-2), alpha-1-antichymotrypsin, and alpha-1-acid glycoprotein emerged from the computational experiments and the outcomes from the promising interaction with COX-2 were confirmed by Western blot, highlighting the reliability of IVS in the field of the natural products.

Chemical Profile, In Vitro Biological Activity and Comparison of Essential Oils from Fresh and Dried Flowers of Lavandula angustifolia L.

The chemical composition of essential oils (EOs) from dried and fresh flowers of Lavandula angustifolia L. (lavender), named LA 2019 and LA 2020, respectively, grown in central Italy was analyzed and compared by GC and GC-MS. For both samples, 61 compounds were identified, corresponding to 97.9% and 98.1% of the total essential oils. Explorative data analysis, performed to compare the statistical composition of the samples, resulted in a high level of global similarity (around 93%). The compositions of both samples were characterized by 10 major compounds, with a predominance of Linalool (35.3-36.0%), Borneol (15.6-19.4%) and 1,8-Cineole (11.0-9.0%). The in vitro antibacterial activity assay by disk diffusion tests against Bacillus subtilis PY79 and Escherichia coli DH5α showed inhibition of growth in both indicator strains. In addition, plate counts revealed a bactericidal effect on E. coli, which was particularly noticeable when using oil from the fresh lavender flowers at the highest concentrations. An in vitro antifungal assay showed that the EOs inhibited the growth of Sclerotium rolfsii, a phytopathogenic fungus that causes post-harvest diseases in many fruits and vegetables. The antioxidant activity was also assessed using the ABTS free radical scavenging assay, which showed a different antioxidant activity in both EOs. In addition, the potential application of EOs as a green method to control biodeterioration phenomena on an artistic wood painting (XIX century) was evaluated.

Metabolite variation in three edible Italian Allium cepa L. by NMR-based metabolomics: a comparative study in fresh and stored bulbs.

INTRODUCTION: In fruits and vegetables, comparative analysis of metabolic plant profiles has a high potential for quality control of active components. Onion (Allium cepa L.) is used fresh or stored as food, spice, and in traditional medicine. Its metabolic content, often with nutraceutical value, makes its level an important factor in agronomic production. OBJECTIVE: To describe for the first time the metabolome of "San Pietro" white onion (WP), and compare its chemical profile with the red onion var. Tropea (RT) and the yellow onion var. Montoro (CM). Furthermore, we also aim to obtain a multivariate model based on NMR fingerprints to discriminate the three Italian A. cepa L. cultivars. METHODS: For the chemical fingerprinting we used NMR-based metabolomics. We investigated the aqueous and chloroform extracts of fresh onion at harvesting time, and after 9-month storage. Principal component analysis (PCA), Partial least squares discriminant analysis (PLS-DA) and Orthogonal partial least squares (OPLS-DA) were used to build reliable models. RESULTS: We obtained a clear discrimination of A. cepa L. varieties for the fresh and stored batches. The statistical model highlighted higher levels of fructo-oligosaccharides (FOS) in the fresh WP; RT showed a high content of glucose, citrate and amino acids, while CM had many sulfur components. In the stored samples (CMS, RTS), carbohydrates and sulfur components decreased, while in WPS the free monosaccharides concentration increased. Linoleic acid was overexpressed in the apolar extracts of CMF and WPF cultivars. CONCLUSION: Metabolomics allows a reliable differentiation among onion varieties, and highlights the potential of fingerprinting for food authentication purposes.

Wound healing activity and phytochemical screening of purified fractions of Sempervivum tectorum L. leaves on HCT 116.

INTRODUCTION: Sempervivum tectorum L. (Crassulaceae), is a succulent perennial plant widespread in Mediterranean countries and commonly used in traditional medicine for ear inflammation, ulcers and skin rashes as a refrigerant and astringent. OBJECTIVE: To demonstrate the therapeutic effects of the plant, various fractions were purified and characterised. The potential wound healing activity, proliferation rate and intracellular signalling cascades were investigated by using human epithelial colorectal carcinoma (HCT 116) cells. METHODOLOGY: An extraction method without organic solvents was applied for the first time. The purification was carried out by droplet counter current chromatography (DCCC) coupled with high-performance liquid chromatography (HPLC) and electrospray ionisation mass spectrometry (ESI-MS) data. By nuclear magnetic resonance (NMR) [1 H, 13 C and two-dimensional (2D) experiments] pure components were identified. Wound healing and cell proliferation assays were utilised to determine the role of the isolated S. tectorum (SVT) fraction on cellular migration and proliferation. The signalling pathways elicited from the SVT fractions, were analysed by Western blot analysis. RESULTS: In this study two rare natural components were identified, namely monosaccharide sedoheptulose and polyalcohol 2-C-methyl-D-erythritol, along with known organic acids and flavonoids. The fractions with high level of sedoheptulose enhance the proliferation and the cellular migration of epithelial HCT 116 cells. The intracellular signalling cascades elicited from the purified fractions induce the c-Src-mediated transactivation of EGFR and the activation of the STAT3 pathway which, in turn, are crucially involved in the cellular proliferation and migration. CONCLUSIONS: Our study demonstrates the efficacy of purified fractions of S. tectorum L. in enhancing cellular proliferation and migration, suggesting their potential role as topical therapeutic treatments for wound healing.

Neuroprotective effects of quercetin 4'-O-ß-d-diglucoside on human striatal precursor cells in nutrient deprivation condition.

Several investigations have demonstrated neuroprotective effects of quercetin, a polyphenol widely present in nature, against neurotoxic chemicals, as well as in neuronal injury/neurodegenerative disease models. Most of these studies have been performed with quercetin aglycone and its metabolites, while scanty data are available on its glycosides. This study is aimed at investigating the neuroprotective effects of quercetin 3,4'-O-ß-d-diglucoside (Q3,4'dG), isolated from the bulbs of the white cultivar (Allium cepa L.), using an in vitro model of human striatal precursor cells (HSPs), a primary culture isolated from the striatal primordium and previously characterized. To study the effect of Q3,4'dG on cell survival, HSPs were exposed to nutrient deprivation created by replacing culture medium with phosphate buffer saline (PBS). Our findings showed that Q3,4'dG treatment significantly promoted cell survival and strongly decreased apoptosis induced by nutrient deprivation, as evaluated by cell proliferation/death analyses. In addition, since the adhesive capacities of cells are essential for cell survival, the expression of some adhesion molecules, such as pancadherin and focal adhesion kinase, was evaluated. Interestingly, PBS exposure significantly decreased the expression of both molecules, while in the presence of Q3,4'dG this effect was prevented. This study provides evidence of a neuroprotective role exerted by Q3,4'dG and suggests its possible implication in sustaining neuronal survival for prevention and treatment of neurodegenerative disorders.

Antioxidant activity and chemical components as potential anticancer agents in the olive leaf (Olea europaea L. cv Leccino.) decoction.

Epidemiological studies have shown that a reduced risk of chronic diseases such as cancer and cardiovascular diseases is correlated with a regular consumption of fruits and vegetable, many of which are rich in polyphenols. The additive and synergistic effect of phytochemicals in fruits and vegetables may reduce chronic diseases related to oxidative stress in human body. Olea europaea L. leaf are rich in phenolic components, which have been proposed to play a role in cancer prevention. The purpose of this study was to identify the main components in the Olea europaea L. leaf (cv. Leccino) preserved during the decoction preparation, in order to delineate the antioxidant activities of the crude extracts and its isolated compounds by using different in vitro assays including DPPH radicalscavenging capacity, total antioxidant capacity (TAC), xanthine oxidase (XO) inhibitory effect and the ability to delay the linoleic acid peroxidation process (ALP). The aqueous decoction was partitioned obtaining four extracts and the n-butanol extract showed the highest antioxidant activity and the highest total phenolic content. Phytochemical investigation leads to the isolation of thirteen secondary metabolites including simple phenolics, flavonoids, secoiridoids whose structures were elucidated by spectroscopic data (1D and 2D NMR) and spectrometric techniques. A significant free radical scavenging effect against DPPH has been evidenced in fraxamoside (1) (EC50 62.6 µM) and taxifolin (5) (EC50 50.0 µM), isolated for the first time from the water decoction. The most active compound in the TAC evaluation, was the 3,4 dihydro-phenyl glycol (8) (0.90 caffeic acid equiv.) while taxifolin and fraxamoside resulted as the most efficient inhibitors of XO activity (IC50 2.7 and 5.2 µM, respectively). Secoxyloganin (4), oleuropein (2) and tyrosol (6) showed the highest ALP activity. This study adds to the growing body of data supporting the bioactivities of phytochemicals and their potential impact on human health.

Novel steroidal components from the underground parts of Ruscus aculeatus L.

Two new furostanol saponins 1–2 and three new sulphated glycosides 3a,b and 4 were isolated from the underground parts of Ruscus aculeatus L., along with four known furostanol and one spirostanol saponins 5–9 and three free sterols. All of the structures have been elucidated on the basis of spectroscopic data 1D and 2D NMR experiments, MS spectra and GC analyses.

Imbricatolic acid from Juniperus communis L. prevents cell cycle progression in CaLu-6 cells.

Imbricatolic acid was isolated from the methanolic extract of the fresh ripe berries of Juniperus communis (Cupressaceae) together with sixteen known compounds and a new dihydrobenzofuran lignan glycoside named juniperoside A. Their structures were determined by spectroscopic methods and by comparison with the spectral data reported in literature. Imbricatolic acid was evaluated for its ability to prevent cell cycle progression in p53-null CaLu-6 cells. This compound induces the upregulation of cyclin-dependent kinase inhibitors and their accumulation in the G1 phase of the cell cycle, as well as the degradation of cyclins A, D1, and E1. Furthermore, no significant imbricatolic acid-induced apoptosis was observed. Therefore, this plant-derived compound may play a role in the control of cell cycle.

Phenolic compounds from Achillea millefolium L. and their bioactivity.

Since antiquity, Achillea millefolium L. (Asteraceae) has been used in traditional medicine of several cultures, from Europe to Asia. Its richness in bioactive compounds contributes to a wide range of medicinal properties. In this study, we assessed A. millefolium methanolic extract and its isolated components for free radical scavenging activity against 2,2-diphenyl-pycrilhydrazyl, total antioxidant capacity (based on the reduction of Cu(++) to Cu(+)), and ability to inhibit lipid peroxidation. The activity against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum was also tested. Chlorogenic acid, its derivatives and some flavonoids isolated by semipreparative HPLC and identified by NMR and spectrometric techniques were the major bioactive constituents of the methanolic extract. The latter exhibited significant antioxidant properties, as well as its flavonol glycosides and chlorogenic acids. With regard to the antiplasmodial activity, apigenin 7-glucoside was the most effective compound, followed by luteolin 7-glucoside, whereas chlorogenic acids were completely inactive. On the whole, our results confirmed A. millefolium as an important source of bioactive metabolites, justifying its pharmaceutical and ethnobotanical use.

Lauroside B, a megastigmane glycoside from Laurus nobilis (bay laurel) leaves, induces apoptosis in human melanoma cell lines by inhibiting NF-κB activation.

Malignant melanoma is a highly aggressive tumor that frequently resists chemotherapy, so the search for new agents for its treatment is of great importance. In the present study, the antiproliferative propensity against human melanoma cell lines of lauroside B (1), a megastigmane glycoside isolated from Laurus nobilis (bay laurel) leaves, was investigated. This compound suppressed the proliferation of three human melanoma cell lines, namely, A375, WM115, and SK-Mel-28. The 1-induced inhibition of human melanoma cell proliferation was due to the induction of apoptosis, as demonstrated by FACS analysis with annexin V/PI staining and confirmed by activation of caspase-3 and by the cleavage of poly(ADP-ribose) polymerase (PARP). Growing evidence implicates NF-κB as an important contributor to metastasis and increased chemoresistance of melanoma. Thus, it was hypothesized that 1-induced apoptosis could be associated with suppression of NF-κB activation. The results showed that exposure of human melanoma cells to 1 inhibited IκB-α degradation and constitutive NF-κB DNA-binding activity as well as the expression, regulated by NF-κB, of two antiapoptotic genes, XIAP and c-FLIP. Induction of apoptosis by 1 in human aggressive melanoma cell lines has a potential high biological value.

Identification of minor secondary metabolites from the latex of Croton lechleri (Muell-Arg) and evaluation of their antioxidant activity.

Dragon's blood (Sangre de drago), a viscous red sap derived from Croton lechleri Muell-Arg (Euphorbiaceae), is extensively used by indigenous cultures of the Amazonian basin for its wound healing properties. The aim of this study was to identify the minor secondary metabolites and test the antioxidant activity of this sustance. A bioguided fractionation of the n-hexane, chloroform, n-butanol, and aqueous extracts led to the isolation of 15 compounds: three megastigmanes, four flavan-3-ols, three phenylpropanoids, three lignans, a clerodane, and the alkaloid taspine. In addition to these known molecules, six compounds were isolated and identified for the first time in the latex: blumenol B, blumenol C, 4,5-dihydroblumenol A, erythro-guaiacyl-glyceryl-beta-O-4'- dihydroconiferyl ether, 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane-1,3-diol and floribundic acid glucoside. Combinations of spectroscopic methods ((1)H-, (13)C- NMR and 2D-NMR experiments), ESI-MS, and literature comparisons were used for compound identification. In vitro antioxidant activities were assessed by DPPH, total antioxidant capacity and lipid peroxidation assays. Flavan-3-ols derivatives (as major phenolic compounds in the latex) exhibited the highest antioxidant activity.

Phenolic glycosides from Foeniculum vulgare fruit and evaluation of antioxidative activity.

Two diglucoside stilbene trimers and a benzoisofuranone derivative were isolated from Foeniculum vulgare fruit together with nine known compounds. Their structures were elucidated by spectral methods including 1D, 2D NMR and MS and chemical methods. Antioxidant activity was tested using three methods: DPPH(), total antioxidant capacity and assay of lipid peroxidation.

New constituents of sweet Capsicum annuum L. fruits and evaluation of their biological activity.

Four new acyclic diterpene glycosides named capsianosides (1-4), together with 12 known compounds, were isolated from the fresh sweet pepper fruits of Capsicum annuum L., a plant used as a vegetable food, spice, and external medicine. The chemical structures of new natural compounds, as well as their absolute configurations, were established by means of spectroscopic data including infrared, high-resolution mass spectrometry, and one- and two-dimensional nuclear magnetic resonance and by chemical derivatization. The known capsidiol (11) showed bacteriostatic properties in vitro against Helicobacter pylori with a minimum inhibitory concentration (MIC) of 200 microg/mL when compared with the commercial drug metronidazole (MIC, 250 microg/mL). Some purified components were also tested for their antioxidant activities.