No Evidence Was Found for the Presence of Terreolides, Terreumols or Saponaceolides H-S in the Fruiting Bodies of Tricholoma terreum (Basidiomycota, Agaricales).

Two different collections of the gilled wild fungus Tricholoma terreum, collected in Italy, were subjected to phytochemical analysis. The fungal material was confidently identified by analysis of the ITS genomic sequences. Using both HR-LC-MS and NMR techniques, no evidence was found for the presence in the fruiting bodies of terreolides, terreumols or saponaceolides H-S, in striking contrast with the isolation of these terpenoids by Chinese authors from a mushroom collected in France and identified as T. terreum. The main cytotoxic terpenoid identified and isolated from the extracts of the specimens investigated in this work was the C30 derivative saponaceolide B, which had been previously isolated from T. saponaceum and other T. terreum collections. Although saponaceolide B is a rather labile molecule, easily degradable by heat or in acidic conditions, our study indicated that none of the extraction protocols used produced saponaceolide H-S or terreolide/terreumol derivatives, thus excluding the possibility that the latter compounds could be extraction artifacts. Considered together, these findings point to the need for the unambiguous identification of mushroom species belonging to the complex genus Tricholoma, characterized by high variability in the composition of metabolites. Moreover, based on our data, T. terreum must be considered an edible mushroom.

A Study of Metabolites from Basidiomycota and Their Activities against Pseudomonas aeruginosa.

The World Health Organization (WHO) promotes research aimed at developing new drugs from natural compounds. Fungi are important producers of bioactive molecules, and they are often effective against other fungi and/or bacteria and are thus a potential source of new antibiotics. Basidiomycota crude extracts, which have previously been proven to be active against Pseudomonas aeruginosa ATCC27853, were subjected to liquid chromatographic separation by RP-18, leading to six macro-fractions for each fungal extract. The various fractions were tested for their bioactivities against P. aeruginosa ATCC27853, and ten of them were characterized by HPLC-HRMS and NMR. Further chromatographic separations were performed for a few selected macro-fractions, yielding seven pure compounds. Bioactivity was mainly found in the lipophilic fractions containing fatty acids and their derivatives, such as hydroxy or keto C-18 unsaturated acids, and in various complex lipids, such as glycolipids and related compounds. More hydrophilic molecules, such as GABA, phenethylamine, two chromogenic anthraquinoids and pistillarin, were also isolated, and their antibacterial activities were recorded. The novelties of this research are as follows: (i) the genera Cortinarius and Mycena have never been investigated before for the synthesis of antibiotic compounds; (ii) the molecules produced by these genera are known, but their production has never been reported in the investigated fungi; (iii) the determination of bacterial siderophore synthesis inhibition by certain compounds from Cortinarius and Mycena.

Cannabis-like activity of Zornia latifolia Sm. detected in vitro on rat cortical neurons: major role of the flavone syzalterin.

Zornia latifolia is a plant suspected to possess psychoactive properties and marketed as a marijuana substitute under the name 'maconha brava'. In this study, the effects of fractions obtained from a 2-propanol extract of aerial portions of the plant were determined by multielectrode array (MEA) analyses on cultured networks of rat cortical neurons. Lipophilic (ZL_lipo, mainly containing flavonoid aglycones), and hydrophilic (ZL_hydro, mainly containing flavonoid glycosides) fractions were initially obtained from the raw extract. ZL_lipo significantly inhibited mean firing rate (MFR) and mean bursting rate (MBR) of MEA recordings, while ZL_hydro induced no inhibition. Column chromatography separation of ZL_lipo yielded five fractions (ZL1-ZL5), among which ZL1 induced the strongest MFR and MBR inhibition. NMR and HPLC-MS analyses of ZL1 revealed the prevalence of the common flavonoids genistein (1) and apigenin (2) (in about a 1:1 ratio), and the presence of the rare flavone syzalterin (6,8-dimethylapigenin) (3) as a minor compound. Exposures of MEA to apigenin and genistein standards did not induce the MFR and MBR inhibition observed with ZL1, whereas exposure to syzalterin standard or to a 1:9 mixture syzalterin-genistein induced effects similar to ZL1. These inhibitory effects were comparable to that observed with high-THC hashish, possibly accounting for the plant psychoactive properties. Data indicate that Z. latifolia, currently marketed as a free herbal product, should be subjected to measures of control. In addition, syzalterin showed distinctive pharmacological properties, opening the way to its possible exploitation as a neuroactive drug.

A Systematic Study of the Antibacterial Activity of Basidiomycota Crude Extracts.

The excessive consumption of antibiotics in clinical, veterinary and agricultural fields has resulted in tremendous flow of antibiotics into the environment. This has led to enormous selective pressures driving the evolution of antimicrobial resistance genes in pathogenic and commensal bacteria. In this context, the World Health Organization (WHO) has promoted research aiming to develop medical features using natural products that are often competitive with synthetic drugs in clinical performance. Fungi are considered an important source of bioactive molecules, often effective against other fungi and/or bacteria, and thus are potential candidates in the search of new antibiotics. Fruiting bodies of sixteen different fungal species of Basidiomycota were collected in the Italian Alps. The identification of fungal species was performed through Internal Transcribed Spacer (ITS) sequencing. Most species belong to genera Cortinarius, Mycena and Ramaria, whose metabolite contents has been scarcely investigated so far. The crude extracts obtained from the above mushrooms were tested for their inhibition activity against five human pathogens: Candida albicans ATCC 14053, C. glabrata ATCC 15126, Staphylococcus aureus NCTC 6571, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumoniae ATCC 13883. Twelve crude extracts showed activity against P. aeruginosa ATCC 27853. Highest activity was shown by some Cortinarius species, as C. nanceiensis.

New acetylenic metabolites from the toxic mushroom Tricholoma pardinum.

Four unprecedented acetylenic alcohols, (Z)-non-7-en-5-yn-1,2,4-triol (1), (Z)-non-7-en-5-yn-1,4-diol (2), (Z)-1,2-dihydroxynon-7-en-5-yn-4-one (3), and (Z)-1-hydroxynon-7-en-5-yn-4-one (4) were isolated from the poisonous mushroom Tricholoma pardinum (Agaricales, Basidiomycota), together with the known compounds 1H-indole-3-carbaldehyde (5) and 6-hydroxy-1H-indole-3-carbaldehyde (6). Their structures were determined by NMR and IR spectroscopy, and mass spectrometry. The crude acetone extract of the mushroom showed potent anti-arthropod activity against Tetranychus urticae (Acarinae), a dangerous crop pest.[Figure: see text].

Phytochemistry of Verbascum Species Growing in Iraqi Kurdistan and Bioactive Iridoids from the Flowers of Verbascum calvum.

Traditional medicine is still widely practiced in Iraqi Kurdistan, especially by people living in villages on mountainous regions; medicinal plants are also sold in the markets of the large towns, such as at Erbil, the capital of the Kurdistan Autonomous Region. About a dozen of Verbascum species (Scrophulariaceae) are commonly employed in the Kurdish traditional medicine, especially for treating burns and other skin diseases. However, the isolation of bioactive secondary metabolites from these plants has not been the subject of intense scientific investigations in Iraq. Therefore, the information reported in the literature about the species growing in Kurdistan has been summarized in the first part of this paper, although investigations have been performed on vegetable samples collected in neighbouring countries, such as Turkey and Iran. In the second part of the work, we have investigated, for the first time, the contents of a methanol and a hydromethanol extract of V. calvum flowers. The extracts exhibited weak antimicrobial activities, whereas the methanol extract showed significant antiproliferative effects against an A549 lung cancer cell line. Moreover, both extracts exhibited a significant dose-dependent free radical scavenging action against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, comparable to that of ascorbic acid. In the subsequent phytochemical study, a high phenolic content was determined in both extracts by the Folin-Ciocalteu assay and medium-pressure liquid chromatographic (MPLC) separation led to the isolation of iridoid glucosides ajugol and aucubin from the methanol extract. In conclusion, the high anti-inflammatory effects of aucubin and the remarkable antioxidant (antiradical) properties of the extracts give scientific support to the traditional use of V. calvum flowers for the preparation in Kurdistan of remedies to cure skin burns and inflammations.

Bioactive Phytochemical Constituents of Wild Edible Mushrooms from Southeast Asia.

Mushrooms have a long history of uses for their medicinal and nutritional properties. They have been consumed by people for thousands of years. Edible mushrooms are collected in the wild or cultivated worldwide. Recently, mushroom extracts and their secondary metabolites have acquired considerable attention due to their biological effects, which include antioxidant, antimicrobial, anti-cancer, anti-inflammatory, anti-obesity, and immunomodulatory activities. Thus, in addition to phytochemists, nutritionists and consumers are now deeply interested in the phytochemical constituents of mushrooms, which provide beneficial effects to humans in terms of health promotion and reduction of disease-related risks. In recent years, scientific reports on the nutritional, phytochemical and pharmacological properties of mushroom have been overwhelming. However, the bioactive compounds and biological properties of wild edible mushrooms growing in Southeast Asian countries have been rarely described. In this review, the bioactive compounds isolated from 25 selected wild edible mushrooms growing in Southeast Asia have been reviewed, together with their biological activities. Phytoconstituents with antioxidant and antimicrobial activities have been highlighted. Several evidences indicate that mushrooms are good sources for natural antioxidants and antimicrobial agents.

Posidonia oceanica (L.) Delile Ethanolic Extract Modulates Cell Activities with Skin Health Applications.

Seagrasses are high plants sharing adaptive metabolic features with both terrestrial plants and marine algae, resulting in a phytocomplex possibly endowed with interesting biological properties. The aim of this study is to evaluate the in vitro activities on skin cells of an ethanolic extract obtained from the leaves of Posidonia oceanica (L.) Delile, family Potamogetonaceae, herein named Posidonia ethanolic extract (PEE). PEE showed high radical scavenging activity, high phenolic content, and resulted rich in chicoric acid, as determined through HPLC-MS analysis. The use of MTT assay on fibroblasts showed a PEE cytotoxicity threshold (IC05) of 50 µg/mL at 48 h, while a sub-toxic dose of 20 µg/mL induced a significant increase of fibroblast growth rate after 10 days. In addition, an ELISA assay revealed that PEE doses of 5 and 10 µg/mL induced collagen production in fibroblasts. PEE induced dose-dependent mushroom tyrosinase inhibition, up to about 45% inhibition at 1000 µg/mL, while 50% reduction of melanin was observed in melanoma cells exposed to 50 µg/mL PEE. Finally, PEE lipolytic activity was assessed by measuring glycerol release from adipocytes following triglyceride degradation. In conclusion, we have collected new data about the biological activities of the phytocomplex of P. oceanica seagrass on skin cells. Our findings indicate that PEE could be profitably used in the development of products for skin aging, undesired hyperpigmentation, and cellulite.

The bioactivity of Hedysarum coronarium extracts on skin enzymes and cells correlates with phenolic content.

CONTEXT: The search for bioactive compounds from botanical sources is attracting much interest. However, differences in chemical composition may occur within the same species depending on different geographical origins. OBJECTIVES: We evaluated the properties on skin enzymes and cells of extracts from sulla legume crop Hedysarum coronarium L. (Fabaceae), collected at two Italian sites near Pisa and Ventimiglia, for possible dermatological and cosmetic applications. MATERIAL AND METHODS: Plant aerial portions were extracted in MTBE/ethyl acetate/acetone, obtaining two extracts named Pisa sulla extract (PSE) and Ventimiglia sulla extract (VSE). Extracts were subjected to chemical characterization, LC-MS/MS analysis and biological assays. RESULTS: PSE showed stronger antiradical scavenging and higher phenolic and flavonoid contents with respect to VSE. LC-MS/MS analysis revealed similar composition for the two extracts, but PSE was richer in condensed tannins and flavonoids, principally rhoifolin, quercetin, naringenin and derivatives. PSE induced stronger inhibition on collagenase and elastase by in vitro enzyme assays, possibly due to higher levels of condensed tannins and quercetin. ELISA bioassay on human dermal fibroblasts revealed stronger PSE induction of collagen production. Determination of glycerol release from adipocytes disclosed stronger stimulation of lipolysis by PSE, allegedly ascribed to higher charge of quercetin and derivatives. In summary, the higher richness in phenolics of PSE is strictly related to stronger bioactivity. DISCUSSION AND CONCLUSIONS: Data indicate that aerial H. coronarium material is suitable for the development of dermatological and cosmeceutical products, but the geographical origin is an important factor for maximally exploiting the biological properties of this species.

Antiproliferative hydroxy-fatty acids from the fodder legume Stylosanthes guianensis.

Stylosanthes guianensis is a fodder legume native from South America and widely grown worldwide. Dried plant material was purchased on the web and taxonomically identified by light and SEM microscopy, and morphological analysis of plants germinated from seeds. The plant was extracted with dichloromethane:2-propanol (9:1). Bioguided fractionation using calcein-AM cytotoxicity assay on HeLa and A431 tumor cells allowed to isolate a lipophilic fraction, endowed with strong cytotoxicity. By means of 1- and 2-D NMR, HPLC-MS, and HR-ESIMS it could be seen that the fraction was an inseparable mixture of complex lipids, mainly consisting of esterified 3-hydroxy fatty acids. Acidic methanolysis of the mixture yielded 3-OH C10 and C12 carboxylic acids, together with palmitic, stearic, and arachidonic acids. Mass values indicate the presence of dimeric and trimeric combinations of 3-hydroxy, C10/C12 acids, and C16/C18/C20 acids, linked via ester bond. Monomeric hydroxyl-fatty acids were also observed, in particular derivatives of mono-hydroxy and di-hydroxy linolenic, linoleic, and oleic acids. 3-O-acylated, esterified fatty acids are unusual in higher plants, and recall motifs of Gram-negative endotoxin lipid A. These oxylipins are likely to be responsible for the antiproliferative activity of S. guianensis, suggesting possible use of the plant in the development of antitumor drugs.

Moraceae Plants with Tyrosinase Inhibitory Activity: A Review.

Hyperpigmentation is an abnormal darkening of the skin mostly derived from excessive melanin production. It is typical of skin disorders including melasma associated to pregnancy or age, freckles, sun freckles and photoaging, age spots, and actinic keratosis. These conditions can be uncomfortable for aesthetic reasons and specific depigmenting treatment is frequently requested. Tyrosinase (EC 1.14.18.1) is the rate-limiting enzyme of melanin synthesis and the main target of antihyperpigmentation remedies. Much interest is focused on compounds able to inhibit tyrosinase activity, particularly natural products, for which there is an increasing demand in the fields of cosmetics and pharmaceutical applications. This review concerns plants from the Moraceae family that have shown tyrosinase inhibition in vitro, including species from the genera Morus, Artocarpus, Maclura (Cudrania), Broussonetia, Milicia (Chlorophora), and Ficus. Compounds with remarkable tyrosinase inhibitory properties have been isolated from the wood and bark of different species, such as calchones, stilbenoids, flavonoids and diterpenes. Studies of structure-activity relationships have suggested that an unsubstituted resorcinol moiety is important for the acquirement of strong tyrosinase inhibition, but various exceptions have been reported. A few species, such as M. alba, A. heterophyllus, A. incisus, Maclura tricuspidata, and C. excelsa, have also shown inhibition of melanin biosynthesis on cultured melanoma cells. In addition, wood extract and the stilbene artocarpin from A. incisus have induced whitening effects on guinea pig skin, while an extract from A. lakoocha has hindered melanin formation in human volunteers. The complex of data indicates that Moraceae plants deserve attention for the development of natural and semi-synthetic tyrosinase inhibitors able to compete with, or outclass, currently available skin whitening drugs.

A New Iridoid Dimer and Other Constituents from the Traditional Kurdish Plant Pterocephalus nestorianus Nábelek.

Accompanied by other rare compounds, a new iridoid dimer, named kurdnestorianoside (1), showing an unprecedented secologanol configuration, has been isolated for the first time from the Kurdish medicinal plant Pterocephalus nestorianus, which is used in Kurdistan for treating oral diseases and inflammation. The structure of 1 was established from 1D- and 2D-NMR spectroscopic data. Kaempferol 3-O-[3,6-di-O-(E)-p-coumaroyl]-ß-d-glucopyranoside (7) showed a remarkable antiproliferative activity against several human tumor cell lines.

Two New Ergosterol Derivatives from the Basidiomycete Cortinarius glaucopus.

Two new sterols 1 and 2 and five known ones 3 - 7 were isolated for the first time from the fruiting bodies of Cortinarius glaucopus. Their structures were established by 1- and 2D-NMR spectra and HR-FABS-MS. The relative configuration of 1 was firmly determined by comparison of the observed 1 H-1 H couplings and NOESY correlations, with those predicted for the computed geometries of the conformers. Calculations were performed by means of DFT with the B3LYP functional at 6-31 + G(d,p) level of theory, in CHCl3 as the solvent. The structures of the new ergosterol derivatives, called glaucoposterol A (1) and B (2), were thus established as (3S,5R,7R,10R,13R,17R,20S,22R,23R,24R)-5,6-epoxy-3,7,23-trihydroxystrophast-8-en-14-one and (22E,3S,5S,9S,10R,13R,17R,20R,24R)-3,5-dihydroxyergosta-6,8(14),22-trien-15-one, respectively. Moreover, the configuration of known strophasterol C (3) was determined as (3S,5R,6S,7R,10R,13R,17R,20S,22S,24R). Glaucoposterol A (1) and strophasterol C (3) represent the second finding in nature of steroids with the rare strophastane skeleton.

A New Ursane-type Triterpenoid and Other Constituents from the Leaves of Crataegus azarolus var. aronia.

A new ursane-type triterpene acid, named azarolic acid (1), along with four known phenolic compounds and four known triterpene acids, was isolated from the crude EtOAc extract of the leaves of Crataegus azarolus var. aronia L. The structure of 1 was determined from 1D and 2D NMR spectroscopic data. Euscaphic acid showed high anti-vasoconstriction effects on aortic rings, supporting the use of this medicinal plant in cardiovascular disease.

Oleuropein-Enriched Olive Leaf Extract Affects Calcium Dynamics and Impairs Viability of Malignant Mesothelioma Cells.

Malignant mesothelioma is a poor prognosis cancer in urgent need of alternative therapies. Oleuropein, the major phenolic of olive tree (Olea europaea L.), is believed to have therapeutic potentials for various diseases, including tumors. We obtained an oleuropein-enriched fraction, consisting of 60% w/w oleuropein, from olive leaves, and assessed its effects on intracellular Ca(2+) and cell viability in mesothelioma cells. Effects of the oleuropein-enriched fraction on Ca(2+) dynamics and cell viability were studied in the REN mesothelioma cell line, using fura-2 microspectrofluorimetry and MTT assay, respectively. Fura-2-loaded cells, transiently exposed to the oleuropein-enriched fraction, showed dose-dependent transient elevations of cytosolic Ca(2+) concentration ([Ca(2+)]i). Application of standard oleuropein and hydroxytyrosol, and of the inhibitor of low-voltage T-type Ca(2+) channels NNC-55-0396, suggested that the effect is mainly due to oleuropein acting through its hydroxytyrosol moiety on T-type Ca(2+) channels. The oleuropein-enriched fraction and standard oleuropein displayed a significant antiproliferative effect, as measured on REN cells by MTT cell viability assay, with IC50 of 22 µg/mL oleuropein. Data suggest that our oleuropein-enriched fraction from olive leaf extract could have pharmacological application in malignant mesothelioma anticancer therapy, possibly by targeting T-type Ca(2+) channels and thereby dysregulating intracellular Ca(2+) dynamics.

Lactarane sesquiterpenes from the European mushrooms Lactarius aurantiacus, L. subdulcis, and Russula sanguinaria.

The results of the first investigation of secondary metabolites occurring in intact and damaged fruiting bodies of the European mushrooms Lactarius aurantiacus, L. subdulcis, and Russula sanguinaria are reported. The pattern of sesquiterpenes in injured R. sanguinaria is dramatically different from that of Lactarius. The structure of the new furanolactarane 4, isolated from L. aurantiacus, was established on the basis of spectral data.

Keratinocyte wound healing activity of galactoglycerolipids from the fern Ophioglossum vulgatum L.

The genus Ophioglossum consists of ferns with different therapeutic properties, including vulnerary virtues. The species Ophioglossum vulgatum L. is traditionally used on wounds and burns as an ointment, suggesting the occurrence of lipophilic compounds with tissue repair properties. We isolated and characterized a galactosyldiacylglycerol mixture (1), composed mainly of 1,2-di-O-linolenoyl-3-O-ß-D-galactopyranosyl-glycerol, from the frond dichloromethane extract. The wound healing properties of 1 were assessed in vitro on keratinocytes. Scratch wound assays showed increased wound closure rates in keratinocyte monolayers exposed to subtoxic doses, previously determined in cytotoxicity assays. The strongest effect, obtained at a dose of 5 µg/mL, approached that of a platelet lysate used in clinical settings. The use of inhibitors of the main cellular pathways involved in wound repair, revealed important roles for intracellular calcium and the ERK1/2 MAP kinase. Conversely, a PCR array of genes involved in wound healing showed an almost total absence of gene modulation. Taken together, the data suggest that 1 acts through a Ca(2+)-dependent, nongenomic mechanism involving the activation of ERK1/2 MAP kinase. Hence, 1 is a main candidate to explain the wound healing virtues of O. vulgatum ointment, and is proposed as a possible new drug in tissue repair and regenerative medicine.

Cadinane sesquiterpenes from the mushroom Lyophyllum transforme.

Two rare cadinane-type sesquiterpenes, lyophyllone A (1) and lyophyllanetriol A (2), were isolated from the mushroom Lyophyllum transforme. The structures were elucidated on the basis of exhaustive NMR techniques, together with MS, UV-Vis and molecular modelling. The absolute configuration of lyophyllone A was determined by ab initio theoretical CD calculation performed by Density Functional Theory (DFT) using the B3PW91/6-31G(d,p) basis set. The experimental CD were found to be in good agreement with the corresponding population-weighted theoretical CD spectra, allowing for the determination of the absolute stereochemistry of the compound.

Terpenoids from Russula lepida and R. amarissima (Basidiomycota, Russulaceae).

Four aristolane sesquiterpenes were isolated from the fruiting bodies of Russula lepida and R. amarissima, namely (1R,2S)-1,2-dihydroxyaristolone (6), (2S,11S)-2,12-dihydroxy-aristolone (7), (1R,2S,11S)-1,2,12-trihydroxyaristolone (8), (1S,2S,11S)-1,2,12-trihydroxy-aristolone (9). In addition, a seco-cucurbitane triterpene, i.e. 3,4-secocucurbita-4,24E-diene-3-hydroxy-26-carboxylic acid (14) was isolated from both species. The configuration at C-2 of the already known rulepidol (2-hydroxyaristolone, 5) was corrected as S instead of R. Several more aristolane and nardosinane sesquiterpenes, as well as cucurbitane triterpenes, already reported both from European and Chinese samples of R. lepida, were also isolated. Compound 14 showed moderate cell growth inhibitory activity.