Profiling of pyrrolizidine alkaloids using a retronecine-based untargeted metabolomics approach coupled to the quantitation of the retronecine-core in medicinal plants using UHPLC-QTOF.

Pyrrolizidine alkaloids (PA) are secondary metabolites of high toxicological relevance. Several PA quantitative methodologies were developed based on a limited number of certified standards, including time consuming solid phase extraction (SPE) purification steps. Herein, we shed light on the variability of PA in herbal extracts and propose a quantification methodology based on ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) for the evaluation of the total PA content as retronecine-equivalents (RE) directly from crude matrices. Particularly in the focus of the investigation were Alkanna spp. (Boraginaceae), which possess a wide range of pharmaceutical properties. In addition, a comparative PA screening of crude and SPE enriched extracts was performed and PA-containing plants from Fabaceae and Compositae families were included to demonstrate universal applicability. In total, 105 PA were identified using HRMSe experiments, specific MS/MS fragmentation PA patterns, a customized in-house library and literature data. Among them, 18 glycosidic PA derivatives were reported for the first time in literature. Using a hierarchical clustering approach, PA distribution in herbal extracts was shown to be family-dependent and significantly different among species. This was further supported by the results of the total PA concentrations, obtained using a retronecine/heliotridine/internal standard-based targeted UHPLC-HRMS quantification method, which varied from 8.64 ± 0.08-3096.28 ± 273.72 µg RE/g extract dry weight in shoots extracts of Alkanna spp. and leaves extracts of Crotalaria retusa L. respectively. Worth mentioning is that the procedure allowed to quantify PA in Alkanna spp. If the procedure based on 35 specific PA recommended by European regulations had been used, results would have been equal to zero for the four species since none were observed in Alkanna spp. Finally, by combining the RE results with the corresponding dereplication results, a customized correction factor for each extract (ranging from 2.12 to 2.48) was assessed leading to a more accurate estimate of the PA content regardless of the molecular weight of each PA. The present methodology will facilitate PA quantification directly from crude extracts and avoid the underestimation the real PA content due to limited availabilty of authentic reference compounds in botanical extracts used in phytomedicines or food supplements/cosmetics.

Polyphasic Systematics of the Fungicolous Genus Cladobotryum Based on Morphological, Molecular and Metabolomics Data.

(1) Background: Species of the anamorphic genus Cladobotryum, are known for their fungicolous lifestyle, making them important mycopathogens in fungiculture. Many morphological, ecological, and molecular phylogenetic studies of the genus have been done to date, but taxonomic uncertainties and challenges still remain. Fungal secondary metabolites, being vastly diverse, are utilised as an extra tool in fungal systematics. Despite being studied for their potentially bioactive compounds, Cladobotryum species are insufficiently investigated regarding metabolomics. (2) Methods: The aim of this study is the identification of Greek strains of Cladobotryum by integrating morphological data, ITS-based phylogeny, and 1H NMR-based metabolomics into a polyphasic approach. (3) Results: Twenty-three strains, isolated from sporophores of macromycetes inhabiting diverse Greek ecosystems, were morphologically identified as Cladobotryum apiculatum, C. fungicola, C. mycophilum, C. varium, C. verticillatum, and Hypomyces rosellus (anamorph C. dendroides), whereas seven strains, which produced red-pigmented metabolites, presented an ambiguous taxonomic position at the species level. Molecular phylogenetics and metabolomics corroborated the morphological findings. (4) Conclusions: Thorough morphological study, ITS region-based phylogeny, and NMR-based metabolomics contribute complementarily to resolving the genus Cladobotryum systematics.

The Metabolic Profile of Anchusa officinalis L. Differs According to Its Associated Arbuscular Mycorrhizal Fungi.

Anchusa officinalis (L.) interacts with various microorganisms including arbuscular mycorrhizal fungi (AMF). Recently, the AMF Rhizophagus irregularis MUCL 41833 has been shown to modulate the metabolome of A. officinalis. However, little information is available on the impact that different AMF species may have on primary and secondary plant metabolites. In this study, four AMF species belonging to the genus Rhizophagus (R. irregularis MUCL 41833, R. intraradices MUCL 49410, R. clarus MUCL 46238, R. aggregatus MUCL 49408), were evaluated for their potential to modulate A. officinalis metabolome under controlled semi-hydroponic cultivation conditions. An untargeted metabolomic analysis was performed using UHPLC-HRMS followed by a multivariate data analysis. Forty-two compounds were reported to be highly modulated in relation to the different AMF associations. Among them, six new secondary metabolites were tentatively identified including two acetyl- and four malonyl- phenylpropanoid and saponin derivatives, all presenting a common substitution at position C-6 of the glycosidic moiety. In addition, an enhanced accumulation of primary and secondary metabolites was observed for R. irregularis and R. intraradices, showing a stronger effect on A. officinalis metabolome compared to R. clarus and R. aggregatus. Therefore, our data suggest that different AMF species may specifically modulate A. officinalis metabolite production.

Comoclathrin, a novel potent skin-whitening agent produced by endophytic Comoclathris strains associated with Andalusia desert plants.

As part of our screening program for the discovery of molecules of microbial origin with skin-whitening activity, 142 diverse fungal endophytes from a wide variety of Andalusia arid plants were screened, applying the OSMAC approach. The fungal strains CF-090361 and CF-090766, isolated from xerophytic plants, were selected as the most promising, while phylogenetic analysis revealed that both strains could represent a new species within the genus Comoclathris. The effect of different fermentation conditions on the production of tyrosinase inhibitory activity was examined, in order to identify the optimum cultivation conditions. LCMS based metabolomics was applied to determine significant differences between the strains and fermentation conditions, and to identify potential bioactive secondary metabolites. Bioassay-guided purification of the main active components led to the isolation of three new compounds (1-3), along with the known compounds graphostrin B (4) and brevianamide M (5). Compound 1 (Comoclathrin) demonstrated the strongest anti-tyrosinase activity (IC50 0.16 µΜ), which was 90-times higher than kojic acid (IC50 14.07 µΜ) used as positive control. Additionally, comoclathrin showed no significant cytotoxicity against a panel of cancer cell lines (HepG2, A2058, A549, MCF-7 and MIA PaCa-2) and normal BJ fibroblasts. These properties render comoclathrin an excellent development candidate as whitening agent.

A Development Strategy of Tailor-made Natural Deep Eutectic Solvents for the Enhanced Extraction of Hydroxynaphthoquinones from Alkanna tinctoria Roots.

Natural hydroxynaphthoquinone enantiomers (HNQs) are well-described pharmaceutical and cosmeceutical agents especially present in the roots of Alkanna tinctoria (L.) Tausch, a species native to the Mediterranean region. In this work, eco-friendly natural deep eutectic solvents (NaDESs) were developed for the selective extraction of these compounds. An extensive screening was performed using more than sixty tailor-made NaDESs. The impact of the intrinsic physicochemical properties on the HNQs extraction efficiency as well as the specificity towards the different enantiomeric pairs was thoroughly investigated. As a result of a multivariate analysis and of the one factor-a-time solvent optimization, the eutectic mixture composed of levulinic acid and glucose (LeG) using a molar ratio of 5 : 1 (molHBA : molHBD) and 20% of water (w/w) was found as the most appropriate mixture for the highest extraction efficiency of HNQs. Further optimization of the extraction process was attained by response surface methodology, using a temperature of 45 °C, a solid-to-liquid ratio of 30 mg/mL, and an extraction time of 50 min. A maximum extraction output of 41.72 ± 1.04 mg/g was reached for HNQs, comparable to that of the commonly used organic solvents. A solid-phase extraction step was also proposed for the recovery of HNQs and for NaDESs recycling. Our results revealed NaDESs as a highly customizable class of green solvents with remarkable capabilities for the extraction of HNQs.

Elastase inhibitory activity of secondary metabolites from the fungus Virgaria nigra CF-231658.

Three known compounds were isolated from Virgaria nigra CF-231658; 2,7-dihydroxy naphthalene (1), virgaricin B (2) and virgaricin (3). The isolated compounds was obtained from liquid-state and agar-supported fermentation using Amberlite XAD-16 solid-phase extraction during the cultivation step. Their structures were elucidated on the basis of 1D and 2D NMR as well as HRMS spectroscopic analyses. The isolated compounds were examined for their ability to inhibit elastase using normal human diploid fibroblasts. Compound 2 displayed the most potent activity with 76.7 ± 2.12% inhibition of the enzyme activity at 5 µM concentration.

The Arbuscular Mycorrhizal Fungus Rhizophagus irregularis MUCL 41833 Modulates Metabolites Production of Anchusa officinalis L. Under Semi-Hydroponic Cultivation.

Anchusa officinalis is recognized for its therapeutic properties, which are attributed to the production of different metabolites. This plant interacts with various microorganisms, including the root symbiotic arbuscular mycorrhizal fungi (AMF). Whether these fungi play a role in the metabolism of A. officinalis is unknown. In the present study, two independent experiments, associating A. officinalis with the AMF Rhizophagus irregularis MUCL 41833, were conducted in a semi-hydroponic (S-H) cultivation system. The experiments were intended to investigate the primary and secondary metabolites (PMs and SMs, respectively) content of shoots, roots, and exudates of mycorrhized (M) and non-mycorrhized (NM) plants grown 9 (Exp. 1) or 30 (Exp. 2) days in the S-H cultivation system. Differences in the PMs and SMs were evaluated by an untargeted ultrahigh-performance liquid chromatography high-resolution mass spectrometry metabolomics approach combined with multivariate data analysis. Differences in metabolite production were shown in Exp. 1. Volcano-plots analysis revealed a strong upregulation of 10 PMs and 23 SMs. Conversely, in Exp. 2, no significant differences in PMs and SMs were found in shoots or roots between M and NM plants whereas the coumarin scoparone and the furanocoumarin byakangelicin, accumulated in the exudates of the M plants. In Exp. 1, we noticed an enhanced production of PMs, including organic acids and amino acids, with the potential to act as precursors of other amino acids and as building blocks for the production of macromolecules. Similarly, SMs production was significantly affected in Exp 1. In particular, the phenolic compounds derived from the phenylpropanoid pathway. Fifteen di-, tri-, and tetra-meric C6-C3 derivatives of caffeic acid were induced mainly in the roots of M plants, while four oleanane-types saponins were accumulated in the shoots of M plants. Two new salvianolic acid B derivatives and one new rosmarinic acid derivative, all presenting a common substitution pattern (methylation at C-9"' and C-9' and hydroxylation at C-8), were detected in the roots of M plants. The accumulation of diverse compounds observed in colonized plants suggested that AMF have the potential to affect specific plant biosynthetic pathways.

Ochraceopyronide, a Rare α-Pyrone-C-lyxofuranoside from a Soil-Derived Fungus Aspergillus ochraceopetaliformis.

The fungal strain was isolated from a soil sample collected in Giza province, Egypt, and was identified as Aspergillus ochraceopetaliformis based on phenotypic and genotypic data. The ethyl acetate extract of the fungal strain exhibited promising activity levels against several pathogenic test organisms and through a series of 1H NMR guided chromatographic separations, a new α-pyrone-C-lyxofuranoside (1) along with four known compounds (2-5) were isolated. The planar structure of the new metabolite was elucidated by detailed analysis of its 1D/2D NMR and HRMS/IR/UV spectroscopic data, while the relative configuration of the sugar moiety was determined by a combined study of NOESY and coupling constants data, with the aid of theoretical calculations. The structures of the known compounds-isolated for the first time from A. ochraceopetaliformis-were established by comparison of their spectroscopic data with those in the literature. All isolated fungal metabolites were evaluated for their antibacterial and antifungal activities against six Gram-positive and Gram-negative bacteria as well as against three human pathogenic fungi.

Isolation of Volatile Compounds with Repellent Properties against Aedes albopictus (Diptera: Culicidae) Using CPC Technology.

The present work describes the use of Centrifugal Partition Chromatography (CPC) for the bio-guided isolation of repellent active volatile compounds from essential oils. Five essential oils (EOs) obtained from three Pinus and two Juniperus species were initially analyzed by gas chromatography-mass spectrometry (GC/MS) and evaluated for their repellent properties against Aedes albopictus. The essential oil from needles of P. pinea (PPI) presented the higher activity, showing 82.4% repellency at a dose of 0.2 µL/cm2. The above EO, together with the EO from the fruits of J. oxycedrus subsp. deltoides (JOX), were further analyzed by CPC using the biphasic system n-Heptane/ACN/BuOH in ratio 1.6/1.6/0.2 (v/v/v). The analysis of PPI essential oil resulted in the recovery of (-)-limonene, guaiol and simple mixtures of (-)-limonene/ß-pheladrene, while the fractionation of JOX EO led to the recovery of ß-myrcene, germacrene-D, and mixtures of α-pinene/ß-pinene (ratio 70/30) and α-pinene/germacrene D (ratio 65/45). All isolated compounds and recovered mixtures were tested for their repellent activity. From them, (-)-limonene, guaiol, germacrene-D as well the mixtures of (-)-limonene/ß-pheladrene presented significant repellent activity (>97% repellency) against Ae. albopictus. The present methodology could be a valuable tool in the effort to develop potent mosquito repellents which are environmentally friendly.

Endophytic Bacteria From the Roots of the Medicinal Plant Alkanna tinctoria Tausch (Boraginaceae): Exploration of Plant Growth Promoting Properties and Potential Role in the Production of Plant Secondary Metabolites.

Alkannin and shikonin (A/S) are enantiomeric naphthoquinones produced in the roots of certain plants from the Boraginaceae family such as Lithospermum spp. and Alkanna spp. They possess antimicrobial, anti-tumoral and wound healing properties. The production of secondary metabolites by Alkanna tinctoria might be influenced by its endomicrobiome. To study the interaction between this medicinal plant and its bacterial endophytes, we isolated bacteria from the roots of wild growing Alkanna tinctoria collected near to Athens and Thessaloniki in Greece. Representative strains selected by MALDI-TOF mass spectrometry were identified by partial 16S rRNA gene sequence analysis. In total, 197 distinct phylotypes of endophytic bacteria were detected. The most abundant genera recovered were Pseudomonas, Xanthomonas, Variovorax, Bacillus, Inquilinus, Pantoea, and Stenotrophomonas. Several bacteria were then tested in vitro for their plant growth promoting activity and the production of cell-wall degrading enzymes. Strains of Pseudomonas, Pantoea, Bacillus and Inquilinus showed positive plant growth properties whereas those of Bacteroidetes and Rhizobiaceae showed pectinase and cellulase activity in vitro. In addition, bacterial responses to alkannin and shikonin were investigated through resistance assays. Gram negative bacteria were found to be resistant to the antimicrobial properties of A/S, whereas the Gram positives were sensitive. A selection of bacteria was then tested for the ability to induce A/S production in hairy roots culture of A. tinctoria. Four strains belonging to Chitinophaga sp., Allorhizobium sp., Duganella sp., and Micromonospora sp., resulted in significantly more A/S in the hairy roots than the uninoculated control. As these bacteria can produce cell-wall degrading enzymes, we hypothesize that the A/S induction may be related with the plant-bacteria interaction during colonization.

Phytochemical characteristics of bergamot oranges from the Ionian islands of Greece: A multi-analytical approach with emphasis in the distribution of neohesperidose flavanones.

The present study describes the peculiar phytochemical characteristics of bergamots cultivated in distinct islands of the Ionian Sea. Ultrahigh-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) supported by 1 and 2D NMR spectroscopy was used for unambiguous metabolic profiling of albedo, flavedo and juice samples. Profile differences were determined using a multi-analytical clustering approach based on high-performance thin-layer chromatography fingerprints and UHPLC-HRMS data. Finally, a validated HPLC method offering good precision (0.12-0.94%) and accuracy (95.25-103.93%) was proposed for the quantification of the major flavanones. A total of 37 secondary metabolites were characterized belonging to flavonoids, limonoids and coumarins. Their distribution was tissue-dependent and varied significantly from bergamots of other geographical locations. Surprisingly, neoeriocitrin was the major flavanone, reaching 1.69 ± 0.05 g/L in the juice and 5.24 ± 0.12 mg/g in albedo. This is the highest reported amount among Citrus species, rendering Ionian bergamots a promising candidate for novel functional products.

Isoflavonoid Profiling and Estrogen-Like Activity of Four Genista Species from the Greek Flora.

As part of our ongoing research on phytoestrogens, we investigated the phytochemical profile and estrogen-like activities of eight extracts from the aerial parts of four Genista species of Greek flora using estrogen-responsive cell lines. Ethyl acetate and methanolic extracts of G. acanthoclada, G. depressa,G. hassertiana, and G. millii were obtained with accelerated solvent extraction and their phytochemical profiles were compared using ultra-high-performance liquid chromatography-high-resolution mass spectrometry (uHPLC-HRMS). Fourteen isoflavonoids, previously isolated from G. halacsyi, were used as reference standards for their identification in the extracts. Thirteen isoflavonoids were detected in both extracts of G. acanthoclada and G. hassertiana, while fewer and far fewer were detected in G. millii and G. depressa, respectively. The ethyl acetate extracts of G. hassertiana and G. acanthoclada displayed 2.45- and 1.79-fold higher, respectively, estrogen-like agonist activity in Ishikawa cells compared to MCF-7 cells at pharmacologically relevant concentrations. Both these extracts, but not that of G. depressa, contained mono- and di-O-ß-d-glucosides of genistein as well as the aglycone, all three of which are known to display full estrogen-like activity at lower-than-micromolar concentrations. The possibility of using preparations rich in G. hassertiana and/or G. acanthoclada extracts as a potentially safer substitute for low-dose vaginal estrogen for menopausal symptoms is discussed.

Degradation Mechanism of 2,4-Dichlorophenol by Fungi Isolated from Marine Invertebrates.

2,4-Dichlorophenol (2,4-DCP) is a ubiquitous environmental pollutant categorized as a priority pollutant by the United States (US) Environmental Protection Agency, posing adverse health effects on humans and wildlife. Bioremediation is proposed as an eco-friendly, cost-effective alternative to traditional physicochemical remediation techniques. In the present study, fungal strains were isolated from marine invertebrates and tested for their ability to biotransform 2,4-DCP at a concentration of 1 mM. The most competent strains were studied further for the expression of catechol dioxygenase activities and the produced metabolites. One strain, identified as Tritirachium sp., expressed high levels of extracellular catechol 1,2-dioxygenase activity. The same strain also produced a dechlorinated cleavage product of the starting compound, indicating the assimilation of the xenobiotic by the fungus. This work also enriches the knowledge about the mechanisms employed by marine-derived fungi in order to defend themselves against chlorinated xenobiotics.

Biological Evaluation and In Silico Study of Benzoic Acid Derivatives from Bjerkandera adusta Targeting Proteostasis Network Modules.

Abstract: A main cellular functional module that becomes dysfunctional during aging is the proteostasis network. In the present study, we show that benzoic acid derivatives isolated from Bjerkandera adusta promote the activity of the two main protein degradation systems, namely the ubiquitin-proteasome (UPP) and especially the autophagy-lysosome pathway (ALP) in human foreskin fibroblasts. Our findings were further supported by in silico studies, where all compounds were found to be putative binders of both cathepsins B and L. Among them, compound 3 (3-chloro-4-methoxybenzoic acid) showed the most potent interaction with both enzymes, which justifies the strong activation of cathepsins B and L (467.3 ± 3.9%) on cell-based assays. Considering that the activity of both the UPP and ALP pathways decreases with aging, our results suggest that the hydroxybenzoic acid scaffold could be considered as a promising candidate for the development of novel modulators of the proteostasis network, and likely of anti-aging agents.

Screening for tyrosinase inhibitors from actinomycetes; identification of trichostatin derivatives from Streptomyces sp. CA-129531 and scale up production in bioreactor.

In the course of a primary screening of 614 microbial actinomycete extracts for the discovery of tyrosinase inhibitors, the EtOAc extract of the fermentation broth of the strain Streptomyces sp. CA-129531 isolated from a Martinique sample, exhibited in cell free and cell-based assays the most promising activity (IC50 value of 63 µg/mL). Scaled-up production in a bioreactor led to the isolation of one new trichostatic acid analogue, namely trichostatic acid B (1), along with six known trichostatin derivatives (2-7), four diketopiperazines (8-11), two butyrolactones (12-13) and one hydroxamic acid siderophore (14). Among them, trichostatin A (4) showed a Ki value of 6.1 µM and six times stronger anti-tyrosinase activity (IC50 2.18 µΜ) than kojic acid (IC50 14.07 µΜ) used as a positive control. Deoxytrichostatin A (6) displayed also strong inhibitory activity against tyrosinase (IC50 19.18 µΜ). Trichostatin A production in bioreactor started together with the exponential phase of growth (day 4) and the maximum concentration was reached at day 9 (2.67 ± 0.13 µg/mL). Despite the cytotoxicity of some individual components, the EtOAc extract showed no cytotoxic effect on HepG2, A2058, A549, MCF-7 and MIA PaCa-2 cell lines, (IC50 >2.84 mg/mL) and against BG fibroblasts at the concentrations where the whitening effect was exerted, reassuring its safety and great tyrosinase inhibitory potential.

Impact of the Cultivation Technique on the Production of Secondary Metabolites by Chrysosporium lobatum TM-237-S5, Isolated from the Sponge Acanthella cavernosa.

The fungi Chrysosporium lobatum TM-237-S5 was isolated from the sponge Acanthella cavernosa, collected from the mesophotic coral ecosystem of the Red Sea. The strain was cultivated on a potato dextrose agar (PDA) medium, coupling solid-state fermentation and solid-state extraction (SSF/SSE) with a neutral macroreticular polymeric adsorbent XAD Amberlite resin (AMBERLITE XAD1600N). The SSF/SSE lead to high chemodiversity and productivity compared to classical submerged cultivation. Ten phenalenone related compounds were isolated and fully characterized by one-dimensional and two-dimensional NMR and HRMS. Among them, four were found to be new compounds corresponding to isoconiolactone, (-)-peniciphenalenin F, (+)-8-hydroxyscleroderodin, and (+)-8-hydroxysclerodin. It is concluded that SSF/SSE is a powerful strategy, opening a new era for the exploitation of microbial secondary metabolites.

Unraveling the Detoxification Mechanism of 2,4-Dichlorophenol by Marine-Derived Mesophotic Symbiotic Fungi Isolated from Marine Invertebrates.

Chlorophenols (CPs) are environmental pollutants that are produced through various anthropogenic activities and introduced in the environment. Living organisms, including humans, are exposed to these toxic xenobiotics and suffer from adverse health effects. More specifically, 2,4-dichlorophenol (2,4-DCP) is released in high amounts in the environment and has been listed as a priority pollutant by the US Environmental Protection Agency. Bioremediation has been proposed as a sustainable alternative to conventional remediation methods for the detoxification of phenolic compounds. In this work, we studied the potential of fungal strains isolated as symbionts of marine invertebrates from the underexplored mesophotic coral ecosystems. Hence, the unspecific metabolic pathways of these fungal strains are being explored in the present study, using the powerful analytical capabilities of a UHPLC-HRMS/MS. The newly identified 2,4-DCP metabolites add significantly to the knowledge of the transformation of such pollutants by fungi, since such reports are scarce.

Comparative UHPLC-HRMS Profiling, Toxicological Assessment, and Protection Against H2O2-Induced Genotoxicity of Different Parts of Opuntia ficus indica.

Opuntia ficus indica has been an important dietary source and a traditionally used medicinal plant. Given the promising health-promoting properties of this plant, a comparative toxicological assessment and antioxidant bioevaluation of extracts from different parts of the plant were carried out in relation to their chemical profile. Toxicity was examined at multiple endpoints using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Comet and the γH2AX In-Cell Western Assay, while hyphenated ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis was carried out to identify main constituents. None of the extracts showed any cytotoxic and genotoxic effect on cell lines used, apart from the flower extract in HepG2 cells at the highest concentration tested (2.5 mg/mL). Both fruit flesh and seed extracts demonstrated a prominent protective effect against H2O2-induced genotoxicity in almost all concentrations tested, while extracts originated from flowers and cladodes were effective only at the low non-cytotoxic (0.312 and 0.625 mg/mL) and high (1.25 and 2.5 mg/mL) concentrations, respectively. In total, 2 phenolic acids, 12 flavonoids, along with 3 feruloyl derivatives and the plant pigment indicaxanthin, were tentatively identified by UHPLC-HRMS analysis. Phenolic acids (compounds 1 and 2) were mainly distributed in cladodes (64.6%), while flavonoids (3-14) in the flowers (81.8%). Overall, the highest amount of total flavonoids (22.76 ± 0.015 mg of quercetin equivalent [QE]/g) and total phenolics (62.80 ± 0.009 mg gallic acid equivalents [GAE]/g) was found in the flower extract. Flavonoid glycosides have not been detected in the seeds and the flesh, while the fruit seed extract contained mainly feruloyl derivatives. Our data provide convincing evidences for the lack of cytotoxic and genotoxic effects of O. ficus indica aqueous extracts and, in parallel, support the potential for further exploitation of this plant in the food supplement or functional food sector.

Osmanicin, a Polyketide Alkaloid Isolated from Streptomyces osmaniensis CA-244599 Inhibits Elastase in Human Fibroblasts.

The strain Streptomyces osmaniensis CA-244599 isolated from the Comoros islands was submitted to liquid-state fermentation coupled to in situ solid-phase extraction with amberlite XAD-16 resin. Elution of the trapped compounds on the resin beads by ethyl acetate afforded seven metabolites, osmanicin (1), streptazolin (2), streptazone C (3), streptazone B1 (4), streptenol C (5), nocardamine (6) and desmethylenylnocardamine (7). Osmanicin (1) is a newly reported unusual scaffold combining streptazolin (2) and streptazone C (3) through a Diels-Alder type reaction. Experimental evidence excluded the spontaneous formation of 1 from 2 and 3. The isolated compounds were evaluated for their ability to inhibit elastase using normal human diploid fibroblasts. Compound 1 exhibited the most potent activity with an IC50 of 3.7 µM.

Cercospora sp. as a source of anti-aging polyketides targeting 26S proteasome and scale-up production in submerged bioreactor.

From a large screening of microbial extracts for the discovery of proteasome modulating natural products, the fungal strain Cercospora sp. (CF-223709) was selected as the most promising for further investigation. Different liquid cultures of the strain were initially screened for their anti-oxidant activity (DPPH, ABTS) and for their cytotoxicity against the A2058, HepG2 and CCD25sk cell lines. A detailed chemical analysis and evaluation of the capacity to activate 26S-proteasome was followed for the most active extract. Three main polyketides were isolated and characterized by extensive analysis of NMR and HRMS spectra data as penialidine F (1), fulvic acid (2), and SB238569 (3). Fulvic acid showed the most significant anti-oxidant activity. Its IC50 value (8.16 µM) against the ABTS radical resulted 3-fold lower than the standard trolox. Fulvic acid also demonstrated a significant effect on proteasome by enhancing the chymotrypsin- and caspase-like activities of the 26S proteasome of human fibroblasts by 71.43% and 37.5% at 1 µM, respectively. Furthermore by scaling up the culture in a 30 L submerged bioreactor, Cercospora sp. produced up to 162.6 ±â€¯1.3 mg of fulvic acid/L. Our findings suggest that CF-223709 can be a promising source of proteasome activating natural compounds.