Ephedra foeminea as a Novel Source of Antimicrobial and Anti-Biofilm Compounds to Fight Multidrug Resistance Phenotype.

Plants are considered a wealthy resource of novel natural drugs effective in the treatment of multidrug-resistant infections. Here, a bioguided purification of Ephedra foeminea extracts was performed to identify bioactive compounds. The determination of antimicrobial properties was achieved by broth microdilution assays to evaluate minimal inhibitory concentration (MIC) values and by crystal violet staining and confocal laser scanning microscopy analyses (CLSM) to investigate the antibiofilm capacity of the isolated compounds. Assays were performed on a panel of three gram-positive and three gram-negative bacterial strains. Six compounds were isolated from E. foeminea extracts for the first time. They were identified by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) analyses as the well-known monoterpenoid phenols carvacrol and thymol and as four acylated kaempferol glycosides. Among them, the compound kaempferol-3-O-α-L-(2″,4″-di-E-p-coumaroyl)-rhamnopyranoside was found to be endowed with strong antibacterial properties and significant antibiofilm activity against S. aureus bacterial strains. Moreover, molecular docking studies on this compound suggested that the antibacterial activity of the tested ligand against S. aureus strains might be correlated to the inhibition of Sortase A and/or of tyrosyl tRNA synthase. Collectively, the results achieved open interesting perspectives to kaempferol-3-O-α-L-(2″,4″-di-E-p-coumaroyl)-rhamnopyranoside applicability in different fields, such as biomedical applications and biotechnological purposes such as food preservation and active packaging.

In vitro characterization of iridoid and phenylethanoid glycosides from Cistanche phelypaea for nutraceutical and pharmacological applications.

"Desert hyacinths" are a remarkable group of parasitic plants belonging to genus Cistanche, including more than 20 accepted species typically occurring in deserts or coastal dunes parasitizing roots of shrubs. Several Cistanche species have long been a source of traditional herbal medicine or food, being C. deserticola and C. tubulosa the most used in China. This manuscript reports the isolation and identification of some phenylethanoid and iridoid glycosides, obtained from the hydroalcoholic extract of C. phelypaea collected in Spain. The present study aims to characterize the antioxidant activity of C. phelypaea metabolites in the light of their application in nutraceutical and cosmeceutical industries and the effect of acetoside, the most abundant metabolite in C. phelypaea extract, on human keratinocyte and pluripotent stem cell proliferation and differentiation. Our study demonstrated that acetoside, besides its strong antioxidant potential, can preserve the proliferative potential of human basal keratinocytes and the stemness of mesenchymal progenitors necessary for tissue morphogenesis and renewal. Therefore, acetoside can be of practical relevance for the clinical application of human stem cell cultures in tissue engineering and regenerative medicine.

Specialized Metabolites from the Allelopathic Plant Retama raetam as Potential Biopesticides.

To cope with the rising food demand, modern agriculture practices are based on the indiscriminate use of agrochemicals. Although this strategy leads to a temporary solution, it also severely damages the environment, representing a risk to human health. A sustainable alternative to agrochemicals is the use of plant metabolites and plant-based pesticides, known to have minimal environmental impact compared to synthetic pesticides. Retama raetam is a shrub growing in Algeria's desert areas, where it is commonly used in traditional medicine because of its antiseptic and antipyretic properties. Furthermore, its allelopathic features can be exploited to effectively control phytopathogens in the agricultural field. In this study, six compounds belonging to isoflavones and flavones subgroups have been isolated from the R. raetam dichloromethane extract and identified using spectroscopic and optical methods as alpinumisoflavone, hydroxyalpinumisoflavone, laburnetin, licoflavone C, retamasin B, and ephedroidin. Their antifungal activity was evaluated against the fungal phytopathogen Stemphylium vesicarium using a growth inhibition bioassay on PDA plates. Interestingly, the flavonoid laburnetin, the most active metabolite, displayed an inhibitory activity comparable to that exerted by the synthetic fungicide pentachloronitrobenzene, in a ten-fold lower concentration. The allelopathic activity of R. raetam metabolites against parasitic weeds was also investigated using two independent parasitic weed bioassays to discover potential activities on either suicidal stimulation or radicle growth inhibition of broomrapes. In this latter bioassay, ephedroidin strongly inhibited the growth of Orobanche cumana radicles and, therefore, can be proposed as a natural herbicide.

Cytotoxicity and Antiviral Properties of Alkaloids Isolated from Pancratium maritimum.

Ten Amaryllidaceae alkaloids (AAs) were isolated for the first time from Pancratium maritimum collected in Calabria region, Italy. They belong to different subgroups of this family and were identified as lycorine, which is the main alkaloid, 9-O-demethyllycorine, haemanthidine, haemanthamine, 11-hydroxyvittatine, homolycorine, pancracine, obliquine, tazettine and vittatine. Haemanthidine was isolated as a scalar mixture of two 6-epimers, as already known also for other 6-hydroxycrinine alkaloids, but for the first time they were separated as 6,11-O,O'-di-p-bromobenzoyl esters. The evaluation of the cytotoxic and antiviral potentials of all isolated compounds was undertaken. Lycorine and haemanthidine showed cytotoxic activity on Hacat cells and A431 and AGS cancer cells while, pancracine exhibited selective cytotoxicity against A431 cells. We uncovered that in addition to lycorine and haemanthidine, haemanthamine and pancracine also possess antiretroviral abilities, inhibiting pseudotyped human immunodeficiency virus (HIV)−1 with EC50 of 25.3 µM and 18.5 µM respectively. Strikingly, all the AAs isolated from P. maritimum were able to impede dengue virus (DENV) replication (EC50 ranged from 0.34−73.59 µM) at low to non-cytotoxic concentrations (CC50 ranged from 6.25 µM to >100 µM). Haemanthamine (EC50 = 337 nM), pancracine (EC50 = 357 nM) and haemanthidine (EC50 = 476 nM) were the most potent anti-DENV inhibitors. Thus, this study uncovered new antiviral properties of P. maritimum isolated alkaloids, a significant finding that could lead to the development of new therapeutic strategies to fight viral infectious diseases.

Terpestacin, a toxin produced by Phoma exigua var. heteromorpha, the causal agent of a severe foliar disease of oleander (Nerium oleander L.).

Since 1987, several cytochalasins were isolated from Phoma exigua var. heteromorpha, the causal agent of foliar blight disease of oleander (Nerium oleander L.), and chemically and biologically characterised. During the purification process of a large-scale production of cytochalasins A and B, necessary to continue the study on their anticancer activity, a metabolite having a different carbon skeleton compared to that of cytochalasans, was isolated. It was identified as terpestacin, a well-known toxic fungal stestertepenoid, isolated for the first time from P. exigua var. heteromorpha, by spectroscopic investigation (essentially 1D and 2D 1H and 13C-NMR and ESI MS) and optical methods in comparison with the literature data. Terpestacin and some its derivatives (including a natural one, fusaproliferin) were prepared and tested for their biological activity. Terpestacin and fusaproliferin had some inhibitory effects on seed germination of Phelipanche ramosa, whereas none of the compounds caused phytotoxic effects on weed leaves.[Formula: see text].

The Assignment of the Absolute Configuration of Non-Cyclic Sesquiterpenes by Vibrational and Electronic Circular Dichroism: The Example of Chiliadenus lopadusanus Metabolites.

9-Hydroxynerolidol, 9-oxonerolidol, and chiliadenol B are three farnesane-type sesquiterpenoids isolated from Chiliadenus lopadusanus that have shown an interesting activity against human pathogens as Gram+ and Gram- bacteria resistant to antibiotics. However, the absolute configuration (AC) of these interesting sesquiterpenes has not been assigned so far. Vibrational and electronic circular dichroism spectra have been recorded and correlations are pointed out for the three compounds. Density functional theory (DFT) calculations are used in conjunction with Mosher's method of investigation to assign AC. Statistical analysis is considered to quantitatively define the choice of AC from VCD spectra.

Isolation and Biological Characterization of Homoisoflavanoids and the Alkylamide N-p-Coumaroyltyramine from Crinum biflorum Rottb., an Amaryllidaceae Species Collected in Senegal.

Crinum biflorum Rottb. (syn. Crinum distichum) is an Amaryllidaceae plant used in African traditional medicine but very few studies have been performed on this species from a chemical and applicative point of view. Bulbs of C. biflorum, collected in Senegal, were extracted with ethanol by Soxhlet and the corresponding organic extract was purified using chromatographic methods. The pure compounds were chemically characterized by spectroscopic techniques (1D and 2D 1H and 13C NMR, HR MS and ECD) and X-ray analysis. Four homoisoflavonoids (1-4) and one alkylamide (5) were isolated and characterized as 5,6,7-trimethoxy-3-(4-hydroxybenzyl)chroman-4-one (1), as 3-hydroxy-5,6,7-trimethoxy-3-(4-hydroxybenzyl)chroman-4-one (2), as 3-hydroxy-5,6,7-trimethoxy-3-(4-methoxybenzyl)chroman-4-one (3) and as 5,6,7-trimethoxy-3-(4-methoxybenzyl)chroman-4-one (4), and the alkylamide as (E)-N-(4-hydroxyphenethyl)-3-(4-hydroxyphenyl)acrylamide (5), commonly named N-p-coumaroyltyramine. The relative configuration of compound 1 was verified thanks to the X-ray analysis which also allowed us to confirm its racemic nature. The absolute configurations of compounds 2 and 3 were assigned by comparing their ECD spectra with those previously reported for urgineanins A and B. Flavanoids 1, 3 and 4 showed promising anticancer properties being cytotoxic at low micromolar concentrations towards HeLa and A431 human cancer cell lines. The N-p-coumaroyltyramine (5) was selectively toxic to A431 and HeLa cancer cells while it protected immortalized HaCaT cells against oxidative stress induced by hydrogen peroxide. Compounds 1-4 also inhibited acetylcholinesterase activity with compound 3 being the most potent. The anti-amylase and the strong anti-glucosidase activity of compound 5 were confirmed. Our results show that C. biflorum produces compounds of therapeutic interest with anti-diabetic, anti-tumoral and anti-acetylcholinesterase properties.

Pinofuranoxins A and B, Bioactive Trisubstituted Furanones Produced by the Invasive Pathogen Diplodia sapinea.

Two new bioactive trisubstituted furanones, named pinofuranoxins A and B (1 and 2), were isolated from Diplodia sapinea, a worldwide conifer pathogen causing severe disease. Pinofuranoxins A and B were characterized essentially by NMR and HRESIMS spectra, and their relative and absolute configurations were assigned by NOESY experiments and computational analyses of electronic circular dichroism spectra. They induced necrotic lesions on Hedera helix L., Phaseolus vulgaris L., and Quercus ilex L. Compound 1 completely inhibited the growth of Athelia rolfsii and Phytophthora cambivora, while 2 showed antioomycetes activity against P. cambivora. In the Artemia salina assay both toxins showed activity inducing larval mortality.

Sesquiterpene Lactones from Cotula cinerea with Antibiotic Activity against Clinical Isolates of Enterococcus faecalis.

Cotula cinerea, belonging to the tribe Anthemideae, is a plant widespread in the Southern hemisphere. It is frequently used in folk medicine in North African countries for several of its medical properties, shown by its extracts and essential oils. The dichloromethane extract obtained from its aerial parts demonstrated antibiotic activity against Enterococcus faecalis and was fractionated by bioguided purification procedures affording five main sesquiterpene lactones. They were identified by spectroscopic methods (NMR and ESIMS data) as guaiantrienolides, i.e., 6-acetoxy-1ß-,6-acetoxy-1α-, and 6-acetoxy-10-ß-hydroxyguaiantrienolide (1-3), and germacrenolides, i.e., haagenolide and 1,10-epoxyhaagenolide (4 and 5). The absolute configuration was assigned by applying the advanced Mosher's method to haagenolide and by X-ray diffraction analysis to 1,10-epoxyhaagenolide. The specific antibiotic and antibiofilm activities were tested toward the clinical isolates of Enterococcus faecalis. The results showed that compounds 3-5 have antibacterial activity against all the strains of E. faecalis, while compound 2 exhibited activity only toward some strains. Compound 1 did not show this activity but had only antibiofilm properties. Thus, these metabolites have potential as new antibiotics and antibiofilm against drug resistant opportunistic pathogens.

Biodegradable polymers as carriers for tuning the release and improve the herbicidal effectiveness of Dittrichia viscosa plant organic extracts.

BACKGROUND: The organic extracts (OEs) of Dittrichia viscosa, a ruderal plant common in the Mediterranean regions, proved to have herbicidal properties. In order to improve OE effectiveness and to develop novel eco-friendly bioherbicidal products, different amounts of OE were included in poly(butylene succinate)- and polycaprolactone-based films (PBS and PCL, respectively). Particular attention was given to the study of interactions between the polymers and OEs, with a deep spotlight concerning the influence of OEs on structural, morphological and thermal properties of both polymers, in order to assess the OE releasing kinetics from the matrices and its tuned herbicidal action against seeds. RESULTS: The bioassays carried out on Lepidium sativum and Phelipanche ramosa seeds evidenced a more controlled and effective OE release by PBS than PCL, and a longer lasting efficacy by the polymers with a higher OE content. The chemical-physical analyses were performed on films before and after biological assays. The thermogravimetric analysis confirmed that OE was a thermal stabilizer of the polymer; the presence of OE and polymer separated degradative kinetics suggested that only a partial and functional miscibility between polymers and OE occurred. The morphological analysis confirmed the good OE dispersion between PBS and PCL molecular chains. Infrared spectroscopy highlighted the enhanced hydrolysed structure of the doped polymers after the bioassays. These outcomes well matched the quantitative information outlined by release kinetics. DISCUSSION: The use of biodegradable polymers allows the effectiveness and tuning of the release of the formulated bioactive compounds to be improved. The easy-to-obtain and easy-to-formulate OE could become a suitable and environmentally friendly instrument in weed management programmes.

Secondary metabolites of Thymelaea hirsuta, a plant collected from the Sicilian Island of Lampedusa.

An investigation of the secondary metabolites was carried out on Thymelaea hirsuta collected from Lampedusa, the largest island of the Pelagie archipelago, located about 100 km from the North African coast and 200 km from the coast of Sicily. Ten compounds were isolated and found to belong to different classes of natural products as chromenes, cyclohexanones, furanyl, bis-furanyl and furanone polyphenols, and acrylates. Compounds 7, 8, 9 and 10 were slightly phytotoxic to lettuce reaching phytotoxicity of 1 (7, 8 and 9) and 2 (10) using a 1-5 point scale. None of the compounds were active against Agrostis stolonifera L., a perennial grass of the Poaceae family. Tested against three Colletotrichum species (C. acutatum, C. fragarie and C. gloeosporioides) pathogenic for agricultural plants, only compound 6 had activity against all three species, but it was not as active as captan, the commercial fungicide used as a positive control.

α-Costic acid, a plant sesquiterpene with acaricidal activity against Varroa destructor parasitizing the honey bee.

The organic extract of the aerial parts of Dittrichia viscosa, a perennial native plant of the Mediterranean basin, showed a significant acaricidal activity against Varroa destructor, the parasite mite of Apis mellifera, commonly called honey bee. Among the metabolites isolated from the organic extract of this Asteraceae, α-costic acid showed to be one of the compounds responsible for the toxic activity exhibited by the crude plant extract on this parasite mite species. In addition to the toxic effect a clear acaricidal response has been recorded when the parasitic mite was exposed to 1 mg/mL concentration of α-costic acid while no effects have been showed on honey bees using the same compound at the same concentration. This finding suggests a potential use of α-costic acid to control Varroa mites. The possibility to reliably achieve absolute configuration of α-costic acid by DFT computational analysis of chiroptical spectra has been also demonstrated.†.

Advances in the Chemical and Biological Characterization of Amaryllidaceae Alkaloids and Natural Analogues Isolated in the Last Decade.

Amaryllidaceae are bulbous wild and cultivated plants well known for their beautiful flowers and pharmaceutical applications, essentially due to the alkaloids and flavonoids content. Hundreds of alkaloids have been isolated until now and several scientific publications reported their sources, chemical structures, and biological activities. During the last decade, some unstudied Amaryllidaceae plants were the object of in-depth investigations to isolate and chemically and biologically characterize new and already known alkaloids as well as some analogues. This review describes the isolation and chemical and biological characterization of the Amaryllidaceae alkaloids, and their analogues obtained in the last decade, focusing the discussion on the new ones.

Antimicrobial secondary metabolites of an endolichenic Aspergillus niger isolated from lichen thallus of Parmotrema ravum.

A new 6-benzyl-γ-pyrone (1), named aspergyllone was isolated from the culture filtrates of an endolichenic fungus Aspergillus niger Tiegh, obtained from lichen thallus Parmotrema ravum (Krog & Swinscow) Serus, collected in India. 1 was isolated for the first time from an endolichenic fungus together with six other known metabolites identified as aurasperones A (2) and D (3), asperpyrone A (4), fonsecinone A (5), carbonarone A (6) and pyrophen (7). The compounds were tested against a panel of human, plant, food borne and fish pathogens. Aspergyllone showed strong selective antifungal activity against Candida parapsilosis (Ashford) Langeron & Talice, with an IC50 of 52 µg/mL. Aurasperone A and pyrophen showed moderate to strong antimicrobial activity inhibiting seven different test pathogens, being pyrophen active with IC50 ranging from 35 to 97 µg/mL.[Formula: see text].

Inuloxin E, a New Seco-Eudesmanolide Isolated from Dittrichia viscosa, Stimulating Orobanche cumana Seed Germination.

A new sesquiterpenoid belonging to the subgroup seco-eudesmanolides and named inuloxin E was isolated from Dittrichia viscosa, together with the already known sesquiterpenoids inuloxins A-D and α-costic acid. Inuloxin E was characterized by spectroscopic data (essentially NMR and ESI MS) as 3-methylene-6-(1-methyl-4-oxo-pentyl)-3a,4,7,7a-tetrahydro-3H-benzofuran-2-one. Its relative configuration was determined by comparison with the closely related inuloxin D and chemical conversion of inuloxin E into inuloxin D and by the observed significant correlation in the NOESY spectrum. Both inuloxins D and E induced germination of the parasitic weed Orobanche cumana, but were inactive on the seeds of Orobanche minor and Phelipanche ramosa. The germination activity of some hemisynthetic esters of inuloxin D was also investigated.

Absolute configuration assignment to anticancer Amaryllidaceae alkaloid jonquailine.

Jonquailine, a new alkaloid recently isolated from Narcissus jonquilla quail, an Amaryllidaceae species cultivated for its flowers fragrance in Europe and USA, shows very significant anti-proliferative activity against several malignant cancer cell types. Although it was reported that this activity is related to the functionalities and to its stereochemistry at C-8 of B ring, the absolute configuration at this stereocenter was not known. Density functional theory (DFT) calculations of chiroptical properties, namely electronic circular dichroism (ECD), vibrational circular dichroism (VCD), and optical rotatory dispersion (ORD) are employed here to complete assignment of absolute configuration of jonquailine, and then, by extension, to its analogues pretazettine and 8-O-methylpretazettine. While ECD is not discriminating and ORD is of limited use, VCD reveals decisive in the task of absolute configuration assignment.

Antimould microbial and plant metabolites with potential use in intelligent food packaging.

Moulds food infestation is a heavy dangerous problem for human health and also could generate heavy economic losses. The intelligent packaging using eco-friendly biodegradable biofilm incorporating bioactive natural safe compounds represents a new frontier. This manuscript reports the inhibitory activity of 12 bacterial, fungal and plant metabolites against Penicillium roqueforti and Aspergillus niger. Among them α-costic acid and ungeremine (3 and 12) are the most promising as potential biofungicide against both fungal strains. They inhibited fungal growth by more than 60% respect to the control at 72 h and this activity persisted also at 96 h. Ungeremine showed MIC90 lower than 0.003 mg/mL after 48 h of incubation and of 0.025 mg/mL at 72 h against P. roqueforti. The MIC90 value for A. niger was 0.2 mg/mL at 48 h for both compounds. The α-costic acid showed generally MIC values at 48 and 72 h higher than ungeremine.

Sarniensine, a mesembrine-type alkaloid isolated from Nerine sarniensis, an indigenous South African Amaryllidaceae, with larvicidal and adulticidal activities against Aedes aegypti.

A new mesembrine-type alkaloid, named sarniensine, was isolated together with tazettine, lycorine, the main alkaloid, and 3-epimacronine from Nerine sarniensis, with the last two produced for the first time by this plant. This Amaryllidaceae, which is indigenous of South Africa, was investigated for its alkaloid content, because the organic extract of its bulbs showed strong larvicidal activity with an LC50 value of 0.008µgµL-1 against first instar Aedes aegypti larvae and with an LD50 value 4.6µg/mosquito against adult female Ae. aegypti, which is the major vector for dengue, yellow fever and the Zika virus. The extract did not show repellency at MED value of 0.375mgcm2 against adult Ae. aegypti. Sarniensine was characterized using spectroscopic and chiroptical methods as (3aR,4Z,6S,7aS)-6-methoxy-3a-(2'-methoxymethyl-benzo [1,3]dioxol-1'-yl)-1-methyl-2,3,3a,6,7,7a-hexahydro-1H-indole. It was less effective against larva at the lowest concentration of 0.1µgµL-1, however it showed strong adulticidal activity with an LD50 value of 1.38±0.056µgmosquito-1.

Glanduliferins A and B, two new glucosylated steroids from Impatiens glandulifera, with in vitro growth inhibitory activity in human cancer cells.

Impatiens glandulifera has been imported from Himalaya in Europe and is considered as an invasive alien plant whose spreading arouses increasing interest among scientific literature. Via anti-cancer bioguiding, two new glucosylated steroids, named glanduliferins A and B, were isolated from the dried stem of I. glandulifera plants, together with the well-known α-spinasterol and 2-methoxy-1,4-naphthoquinone, which are also isolated from roots and leaves. They were characterized as 17-(2-hydroxy-2-pentamethylcyclopropyl-ethyl)-10,13-dimethyl-2,3,4,5,6,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopents[a]phenathren-3-O-(4-O-acetyl)-α-D-glucopyranoside and 17-(4-ethyl-1,5-dimethyl-hex-2-enyl)-10,13-dimethyl-2,3,4,5,6,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopents[a]phenathren-3-O-(6-O-acetyl)-ß-D-glucopyranoside using various NMR and HRESIMS techniques and chemical methods. In vitro determination of the growth inhibitory activity of the four isolated compounds using the MTT colorimetric assay revealed mean IC50 growth inhibitory value of ~30 µM for glanduliferin A while glanduliferin B and α-spinasterol were poorly active till 100 µM. 2-methoxy-1,4-naphthoquinone revealed to be active in the single micromolar digit range as previously described. Quantitative videomicroscopy analyses of the effects of glanduliferins A and B suggested cytostatic rather than cytotoxic activity in U373 glioblastoma (GBM) cells.

Inhibition of Spore Germination and Appressorium Formation of Rust Species by Plant and Fungal Metabolites.

Eight fungal and plant metabolites belonging to different classes of naturally occurring compounds, a 24-oxa[14]-cytochalasan as cytochalasin B (1), a trisubstituted isocoumarin as 6-hydroxymellein (2), a tetracyclic pimarane diterpene as sphaeropsidin A (3), a chalcone as cavoxin (4), a pentasubstituted benzofuranone as cyclopaldic acid (5), a bicyclic-sesquiterpene as inuloxin A (6), a epipolythiopiperazine as gliotoxin (7) and a cyclohexene epoxide as epiepoformin (8), were tested for their effectiveness in reducing early stages of development of several major rust fungi from the genera Puccinia and Uromyces. Spore germination and appressoria formation were assessed on pre-treated detached leaves, under controlled conditions. Among the various metabolites evaluated, compounds 5 and 8 were the most effective in inhibiting fungal germination and penetration of all rust species studied at values comparable with those obtained by fungicide application, while compound 4 was phytotoxic to plant leaves at any concentration tested.