HPLC, NMR Based Characterization, Antioxidant and Anticancer Activities of Chemical Constituents from Therapeutically Active Fungal Endophytes.

Fungi generate different metabolites some of which are intrinsically bioactive and could therefore serve as templates for drug development. In the current study, six endophytic fungi namely Aspergillus flavus, Aspergillus tubigenesis, Aspergillus oryzae, Penicillium oxalicum, Aspergillus niger and Aspergillus brasiliensis were isolated and identified from the medicinal plant, Silybum marianum. These endophytic fungi were identified through intra transcribed sequence (ITS) gene sequencing. The bioactive potentials of fungal extracts were investigated using several bioassays such as antibacterial activity by well-diffusion, MIC, MBC, anti-biofilm, antioxidant, and haemolysis. The Pseudomonas aeruginosa strain PAO1 was used to determine the antibiofilm activity. The ethyl acetate extract of Aspergillus flavus showed strong to moderate efficacy against Staphylococcus aureus, Escherichia coli, P. aeruginosa, and Bacillus spizizenii. Aspergillus flavus and Aspergillus brasiliensis exhibited significant antibiofilm activity with IC50 at 4.02 and 3.63 mg/ml while A. flavus exhibited maximum antioxidant activity of 50.8%. Based on, HPLC, LC-MS and NMR experiments kojic acid (1) and carbamic acid (methylene-4, 1-phenylene) bis-dimethyl ester (2) were identified from A. flavus. Kojic acid exhibited DPPH free radical scavenging activity with an IC50 value of 99.3 µg/ml and moderate activity against ovarian teratocarcinoma (CH1), colon carcinoma (SW480), and non-small cell lung cancer (A549) cell lines. These findings suggest that endophytic fungi are able produce promising bioactive compounds which deserve further investigation.

Boerelasins A-D, Four Unprecedented Cytochalasins from the Endophytic Fungus Boeremia Exigua.

Four undescribed cytochalasins (1-4) were isolated from the endophytic fungus Boeremia exigua. Structurally, boerelasin A (1) represents the first example of a cytochalasin with a rare 5/5 bicyclic carbon core. Boerelasin B (2) possesses an unprecedented 5/6/5/6/8 pentacyclic ring system. Boerelasin C (3), a derivative from the common biosynthetic intermediate to 1, is a macrocyclic ring-opening cytochalasin, and boerelasin D (4) contains an uncommon six-carbon alkyl acid side chain. The structures were elucidated based on spectroscopic methods, electronic circular dichroism, spin-spin coupling constants, and calculated nuclear magnetic resonance with DP4+ analysis. These compounds exhibited significant cytotoxicity against the tumor cells.

Diverse aspidosperma-type alkaloids from the leaves of Tabernaemontana bovina with anti-hepatoma activity.

Seventeen undescribed Aspidosperma-type alkaloids (ASPs), along with nine known ones were isolated from the leaves of Tabernaemontana bovina. Taberbovermines A and B were assigned to tabersonine-type with a contracted A- and E-ring, respectively. Taberbovermine C was attributed to tabersonine without D ring. These structures of the ASPs were established on the basis of comprehensive spectroscopic data, electronic circular dichroism calculations and X-ray diffraction. The summaries of structure-activity relationship of tabersonine class were discussed based on hepatoma cells screening.

MAPkinases regulate secondary metabolism, sexual development and light dependent cellulase regulation in Trichoderma reesei.

The filamentous fungus Trichoderma reesei is a prolific producer of plant cell wall degrading enzymes, which are regulated in response to diverse environmental signals for optimal adaptation, but also produces a wide array of secondary metabolites. Available carbon source and light are the strongest cues currently known to impact secreted enzyme levels and an interplay with regulation of secondary metabolism became increasingly obvious in recent years. While cellulase regulation is already known to be modulated by different mitogen activated protein kinase (MAPK) pathways, the relevance of the light signal, which is transmitted by this pathway in other fungi as well, is still unknown in T. reesei as are interconnections to secondary metabolism and chemical communication under mating conditions. Here we show that MAPkinases differentially influence cellulase regulation in light and darkness and that the Hog1 homologue TMK3, but not TMK1 or TMK2 are required for the chemotropic response to glucose in T. reesei. Additionally, MAPkinases regulate production of specific secondary metabolites including trichodimerol and bisorbibutenolid, a bioactive compound with cytostatic effect on cancer cells and deterrent effect on larvae, under conditions facilitating mating, which reflects a defect in chemical communication. Strains lacking either of the MAPkinases become female sterile, indicating the conservation of the role of MAPkinases in sexual fertility also in T. reesei. In summary, our findings substantiate the previously detected interconnection of cellulase regulation with regulation of secondary metabolism as well as the involvement of MAPkinases in light dependent gene regulation of cellulase and secondary metabolite genes in fungi.

Yellow Twig (Nauclea orientalis) from Thailand: Strictosamide as the Key Alkaloid of This Plant Species

Comprehensive phytochemical examination from different perspectives using preparative and analytical chromatographic techniques combined with spectroscopic/spectrometric methods of the so-called "yellow twig" Nauclea orientalis (L.) L. (Rubiaceae) led to the identification of 13 tryptamine-derived (=monoterpene-indole) alkaloids. The identified alkaloids comprise strictosamide and four of its glucosidic derivatives, three oxindole derivatives, and five yellow-colored angustine-type aglycones. Qualitative and quantitative HPLC analyses showed the enrichment of strictosamide in all studied organs. Based on these results, we performed metabolomic analyses of monoterpene-indole alkaloids and made a 1H NMR in vitro monitoring of enzymatic deglucosylation of strictosamide. A comparison of the stability of strictosamide and its enantiomer vincoside lactam by theoretical calculations was also performed revealing a slightly higher stability of vincoside lactam. Additionally, we conducted two different anti-feedant assays of strictosamide using larvae of the polyphageous moth Spodoptera littoralis Boisduval. The obtained results indicate that generally two different biosynthetic pathways are most likely responsible for the overall alkaloid composition in this plant. Strictosamide is the key compound in the broader pathway and most likely the source of the identified angustine-type aglycones, which may contribute significantly to the yellow color of the wood. Its cross-organ accumulation makes it likely that strictosamide is not only important as a reservoir for the further biosynthesis, but also acts in the plants' defense strategy.

Cephalotaxine homologous alkaloids from seeds of Cephalotaxus oliveri Mast.

Six undescribed isoquinoline alkaloids, named as cephaloliverines A-F, were isolated from the seeds of Cephalotaxus oliveri. They were identified by NMR and MS spectroscopic data analyses, combined with the time-dependent density functional theory ECD calculation for cephaloliverines A and B and also by X-ray crystal diffraction for cephaloliverine E. Biosynthetic considerations suggest that cephaloliverines A-D are homologous of cephalotaxine-, homoerythrina- and Erythrina-type alkaloids. The performed bioassay revealed no cytotoxic activity against cancer cells and no neuroprotective properties on HEI-OC-1 cells model.

Trimeric and dimeric Aspidosperma-type alkaloids from leaves of Tabernaemontana divaricata 'Dwaft'.

Continued interest in bioactive monoterpenoid indole alkaloids and the purpose to explore the artificial cultivation influence on the chemical composition in the same plant species, 8 undescribed Aspidosperma-type alkaloids including two unprecedented trimers, taberdivarines A-B (1-2), and six new dimers, taberdivarines CH (3-8), together with 9 known bisindoles were isolated from the leaves of Tabernaemontana divaricata 'Dwaft'. Notably, taberdivarines A and B were the first cases of Aspidosperma-Aspidosperma-Aspidosperma-type alkaloids with furan ring linkage patterns of the natural products. Their structures were elucidated by comprehensive spectroscopic analyse. Compounds 1-8 were screened for the cytotoxicity against three human cancer cell lines, SMMC-7721, HT-29 and A549. Among them, Compound 6 exhibited significant activity against three cell lines with IC50 values of 0.30, 0.75 and 3.41 µM, respectively (IC50 = 3.02, 0.14 and 2.23 µM for the positive control, vinorelbine). Compound 1, 3, 4, 6, 7 and 8 also expressed varying degrees of activity. The structure-activity relationships (SARs) of these alkaloids were discussed.

Highly Aromatic Flavan-3-ol Derivatives from Palaeotropical Artocarpus lacucha Buch.-Ham Possess Radical Scavenging and Antiproliferative Properties.

Phytochemical investigation of leaves and stembark of Artocarpus lacucha collected in Thailand resulted in three yet undescribed isomeric flavan-3-ol derivatives (1-3), the four known compounds gambircatechol (4), (+)-catechin (5), (+)-afzelechin (6) and the stilbene oxyresveratrol (7). Compounds 1 to 3 feature 6/6/5/6/5/6 core structures. All structures were deduced by NMR and MS, while density functional theory (DFT) calculations on B3LYP theory level were performed of compounds 1 to 3 to support the stereochemistry in positions 2 and 3 in the C-ring. Possible biosynthetic pathways leading to 4 are discussed. The DPPH assay revealed high radical scavenging activities for 1 (EC50 = 9.4 ± 1.0 µmol mL-1), 2 (12.2 ± 1.1), 3 (10.0 ± 1.5) and 4 (19.0 ± 2.6), remarkably lower than ascorbic acid (EC50 = 34.9) and α-tocopherol (EC50 = 48.6). A cytotoxicity assay revealed moderate but consistent antiproliferative properties of 1 in CH1/PA-1 (ovarian teratocarcinoma) and SW480 (colon carcinoma) cells, with IC50 values of 25 ± 6 and 34 ± 4 µM, respectively, whereas effects in A549 (non-small cell lung cancer) cells were rather negligible. The performed DCFH-DA assay of 1 in the former cell lines confirmed potent antioxidative effects even in the cellular environment.

Cage-Monoterpenoid Quinoline Alkaloids with Neurite Growth Promoting Effects from the Fruits of Melodinus yunnanensis.

Meloyunnanines A-C, three alkaloids with an unprecedented skeleton, were isolated from fruits of Melodinus yunnanensis. The structures featuring a caged-6/6/5/6/5/5 ring system were elucidated by the analysis of comprehensive spectroscopic and X-ray data. Biosynthetically, meloyunnanines A-C were assigned to monoterpenoid quinoline alkaloids (MQAs), derived from monoterpenoid indole alkaloids through oxidation and rearrangement. These compounds together with three known Melodinus MQAs were evaluated for their neurotrophic activity and scandine N4-oxide exhibited significant effect.

The role of PKAc1 in gene regulation and trichodimerol production in Trichoderma reesei.

BACKGROUND: Trichoderma reesei represents a model system for investigation of plant cell wall degradation and its connection to light response. The cyclic adenosine monophosphate pathway (cAMP pathway) plays an important role in both physiological outputs, being crucial for regulation of photoreceptor function as well as for cellulase regulation on different carbon sources. Phosphorylation of photoreceptors and of the carbon catabolite repressor CRE1 was shown in ascomycetes, indicating a relevance of protein kinase A in regulation of the target genes of these transcription factors as well as an impact on regulation of induction specific genes. Moreover, the cAMP pathway impacts growth and development. RESULTS: Here, we investigated gene regulation by the catalytic subunit of protein kinase A (PKAc1) upon growth on cellulose. We found distinct gene sets for regulation upon growth in light and darkness with an overlap of only 13 genes. PKAc1 regulates metabolic genes as well as transport and defense functions. The overlap of gene regulation by PKAc1 with the genes representing the cAMP dependent regulatory output of the photoreceptor ENV1 indicates an involvement of PKA in this pathway, which counteracts its effects by contrasting regulation. Moreover, we found considerable overlap with the gene sets regulated under cellulase inducing conditions and by the carbon catabolite repressor CRE1. Our analysis also showed that PKAc1 regulates the genes of the SOR cluster associated with the biosynthesis of sorbicillinoids. The homologue of gin4, encoding a CAMK type kinase, which is regulated by PKAc1, CRE1 and YPR2 showed a moderate impact on trichodimerol production. We isolated trichodimerol as representative sorbicillin compound and established a method for its quantification in large sample sets using high performance thin layer chromatography (HPTLC), which can be broadly applied for secondary metabolite screening of mutants or different growth conditions. Due to the high expression levels of the SOR cluster under conditions of sexual development we crosschecked the relevance of PKAc1 under these conditions. We could show that PKAc1 impacts biosynthesis of trichodimerol in axenic growth and upon mating. CONCLUSIONS: We conclude that PKAc1 is involved in light dependent regulation of plant cell wall degradation, including carbon catabolite repression as well as secondary metabolism and development in T. reesei.

Four Yellow Monoterpenoid Quinoline Alkaloids from the Stem of Tabernaemontana bovina.

Four unprecedented yellow alkaloids named as taberbovines A-D (1-4) were isolated from stems of Tabernaemontana bovina Lour. Their structures were elucidated by the comprehensive spectroscopic data, computational chemistry, and X-ray crystal diffraction. Alkaloids 1-4 possessed a unique 6/6/5/6/5 ring system and were assigned to monoterpenoid quinolines. Biosynthetically, the isolated alkaloids may be derived from Aspidosperma-type alkaloid by oxidation and rearrangements. Taberbovines A-D exhibited the good inhibitory activities of formation NO in LPS induced RAW264.7 macrophages.