Copper ion-based chemical elicitation induces production of new benzofurans in Anthostomella brabeji, an endophytic fungus of Paepalanthus planifolius.

The present work reports the effects of chemical elicitors and epigenetic modifiers on the production and diversification of secondary metabolites produced by Anthostomella brabeji - an endophytic fungus isolated from Paepalanthus planifolius (Eriocaulaceae). The fungus was cultivated under four different small-scale culture conditions in potato dextrose broth (PDB): PDB (control), PDB + Mg+2, PDB + Cu+2 and PDB + 5-AZA (5-azacytidine). The incorporation of Cu+2 into PDB medium yielded the most promising results as the most significant differences in the metabolic profile of A. brabeji were observed under this condition. The chemical analysis of the PDB + Cu+2 extract resulted in the isolation of seven metabolites, including three new benzofuran derivatives (2, 4 and 6) and four known compounds (1, 3, 5 and 7). The metabolites were tested using the Gram-positive bacterium Staphylococcus aureus, Gram-negative bacteria Salmonella sp. and Escherichia coli, and six yeasts of Candida albicans and non-albicans. The EtOAc extract (PDB + Cu+2), and compounds 1, 2 and 7 exhibited relevant antifungal activity against Candida spp., with minimum inhibitory concentration ranging from 62.5 to 500.0 µg/mL.

Polyketide- and Terpenoid-Derived Metabolites Produced by a Marine-Derived Fungus, Peroneutypa sp.

Bioassay-guided investigation of the EtOAc-soluble extract of a culture of the marine-derived fungus Peroneutypa sp. M16 led to the isolation of seven new polyketide- and terpenoid-derived metabolites (1, 2, 4-8), along with known polyketides (3, 9-13). Structures of compounds 1, 2, and 4-8 were established by analysis of spectroscopic data. Absolute configurations of compounds 1, 2, 4, 6, 7, and 8 were determined by the comparison of experimental ECD spectra with calculated CD data. Compound 5 exhibited moderate antiplasmodial activity against both chloroquine-sensitive and -resistant strains of Plasmodium falciparum.

New Isoquinoline Alkaloids from Paraphaeosphaeria sporulosa F03, a Fungal Endophyte Isolated from Paepalanthus planifolius.

As part of our continuing efforts to discover new bioactive compounds from endophytic fungal sources, we have investigated the extract of the Paraphaeosphaeria sporulosa F03 strain. The study led to the isolation of four new 3-methyl-isoquinoline alkaloids (1:  - 4: ) and four known polyketides (5:  - 8: ). The structures of compounds 1:  - 4: were elucidated by 1D and 2D NMR experiments and HRMS analysis. The absolute configuration of 4: was determined by comparison of its experimental electronic circular dichroism spectrum with calculated data. Compounds 1:  - 4: exhibited antifungal activity with minimal inhibitory concentration values ranging from 6.25 - 50 µg/mL against six Candida species but they did not present any cytotoxic activity against the human tumor cell lines A549 (lung), MCF-7 (breast), and HepG2 (hepatocellular). In addition, compound 4: exhibited antiplasmodial activity in the low micromolar range (IC50 = 4 µM).

The Methionine 549 and Leucine 552 Residues of Friedelin Synthase from Maytenus ilicifolia Are Important for Substrate Binding Specificity.

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and ß-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.

Anthelmintic activity of a nanoformulation based on thiophenes identified in Tagetes patula L. (Asteraceae) against the small ruminant nematode Haemonchus contortus.

The synthesis of thiophenic compounds, previously identified in Tagetes patula, revealed that 4-(5'-(hydroxymethyl)-[2,2'-bithiophene]-5-yl)but-3-yn-1-ol), or simply Thio1, has a pronounced in vitro anthelmintic effect against Haemonchus contortus, showing 100% efficacy in the egg hatch and larval development tests presenting EC50 = 0.1731 mg.mL-1 and EC50 = 0.3243 mg.mL-1, respectively. So, this compound was selected to preparation of a nanostructured formulation to be orally administered to Santa Inês sheep. In general, from the fecal egg count reduction test (FECRT), it was observed that the product kept the parasitic load in the digestive tract of the hosts stable, with eggs per gram of faeces (EPG) counts having a mean value < 3,000 (EPGmean = 2167.1, efficacy = 36,45%), thus protecting the animals from health risks caused by a massive nematode infestation. To better understand the mode of action of this thiophene derivative, in silico molecular modeling studies were carried out with the glutamate-activated chloride channel (GluCl), a well-known molecular target of anthelmintic compounds. Based on the affinity score (GlideScore = -5.7 kcal.mol-1) and the proposed binding mode, Thio1 could be classified as a potential GluCl ligand, justifying the promising results observed in the anthelmintic assays.

Antibacterial activity of prenylated benzopyrans from Peperomia obtusifolia (Piperaceae).

Peperomia obtusifolia is a herbaceous perennial plant native to the Americas reported as a traditional medicine to treat snake bites and as a skin cleanser. The bioassay-guided fractionation of crude extracts from aerial parts of P. obtusifolia against a panel of clinically important fungi and bacteria, showed that hexane and dichloromethane extracts demonstrated selective bacterial inhibition, allowing the isolation of the known compounds peperobtusin A (1), and 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(3"-methyl-2"-butenyl)-2-(4'-methyl-1',3'-pentadienyl)-2H-1-benzopyran-6-carboxylic acid (2) from dichloromethane extract. Compound 2 was active against Gram-positive bacteria including community acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates and an Enterococcus faecalis vancomycin-resistant strain, with minimal inhibitory concentration (MIC) values of 4 µg/mL (10.8 µM) and 8 µg/mL (21.6 µM) respectively. The interaction of compound 2 with the bacterial membrane was demonstrated by means of Zeta potential experiments on S. aureus, then confirming the membrane damage by fluorescent microscopy experiments.

Maytenin Plays a Special Role in the Regulation of the Endophytic Bacillus megaterium in Peritassa campestris Adventitious Roots.

Peritassa campestris (Celastraceae) root bark accumulates potent antitumor quinonemethide triterpenes (QMTs). When grown in their natural habitat, plants of the family Celastraceae produce different QMTs such as celastrol (3) and pristimerin (4). However, when they are inserted in in vitro culture systems, they accumulate maytenin (1) as the main compound. Recently, Bacillus megaterium was detected as an endophytic microorganism (EM) living inside P. campestris roots cultured in vitro. We hypothesized that compound (1) controls EM growth more efficiently, and that the presence of EMs in the root culture causes compound (1) to accumulate. For the first time, this work has explored plant-microorganism interaction in a species of the family Celastraceae by co-culture with an EM. Live endophytic bacteria were used, and QMT accumulation in P. campestris adventitious roots was our main focus. The antimicrobial activity of the main QMTs against endophytic B. megaterium was also evaluated. Our results showed that compound (1) and maytenol (5) were more effective than their precursors QMTs (3) and (4) in controlling the EM. Co-culture of B. megaterium with roots significantly reduced bacterial growth whereas root development remained unaffected. Compound (1) production was 24 times higher after 48 hr in the presence of the highest B. megaterium concentration as compared to the control. Therefore, P. campestris adventitious roots affect the development of the endophyte B. megaterium through production of QMTs, which in turn can modulate production of compound (1).

CYP712K4 Catalyzes the C-29 Oxidation of Friedelin in the Maytenus ilicifolia Quinone Methide Triterpenoid Biosynthesis Pathway.

The native Brazilian plant Maytenus ilicifolia accumulates a set of quinone methide triterpenoids with important pharmacological properties, of which maytenin, pristimerin and celastrol accumulate exclusively in the root bark of this medicinal plant. The first committed step in the quinone methide triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase friedelin synthase (FRS). In this study, we produced heterologous friedelin by the expression of M. ilicifolia FRS in Nicotiana benthamiana leaves and in a Saccharomyces cerevisiae strain engineered using CRISPR/Cas9. Furthermore, friedelin-producing N. benthamiana leaves and S. cerevisiae cells were used for the characterization of CYP712K4, a cytochrome P450 from M. ilicifolia that catalyzes the oxidation of friedelin at the C-29 position, leading to maytenoic acid, an intermediate of the quinone methide triterpenoid biosynthesis pathway. Maytenoic acid produced in N. benthamiana leaves was purified and its structure was confirmed using high-resolution mass spectrometry and nuclear magnetic resonance analysis. The three-step oxidation of friedelin to maytenoic acid by CYP712K4 can be considered as the second step of the quinone methide triterpenoid biosynthesis pathway, and may form the basis for further discovery of the pathway and heterologous production of friedelanes and ultimately quinone methide triterpenoids.

Combined use of tandem mass spectrometry and computational chemistry to study 2H-chromenes from Piper aduncum.

Natural 2H-chromenes were isolated from the crude extract of Piper aduncum (Piperaceae) and analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) applying collision-induced dissociation. Density functional theory (DFT) calculations were used to explain the preferred protonation sites of the 2H-chromenes based on thermochemical parameters, including atomic charges, proton affinity, and gas-phase basicity. After identifying the nucleophilic sites, the pathways were proposed to justify the formation of the diagnostic ions under ESI-MS/MS conditions. The calculated relative energy for each pathway was in good agreement with the energy-resolved plot obtained from ESI-MS/MS data. Moreover, the 2H-chromene underwent proton attachment on the prenyl moiety via a six-membered transition state. This behavior resulted in the formation of a diagnostic ion due to 2-methylpropene loss. These studies provide novel insights into gas-phase dissociation for natural benzopyran compounds, indicating how reactivity is correlated to the intrinsic acid-base equilibrium and structural aspects, including the substitution pattern on the aromatic moiety. Therefore, these results can be applied in the identification of benzopyran derivatives in a variety of biological samples.

Evaluation of Tagetes patula (Asteraceae) as an ecological alternative in the search for natural control of the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The cattle tick Rhipicephalus microplus is a great sanitary problem and causes huge losses to livestock, being a vector of important diseases. The aim of this work was to verify the action of plant-derived material obtained from Tagetes patula on the life cycle of R. microplus, as well as to analyze the action of these extracts on ovary cells of engorged females. In the Adult Immersion Test, the crude ethanolic extract (TpEtOH) showed an efficacy of 99.2% (LC50 = 18.60 mg mL-1). From the repellency test, it was found that both the TpEtOH and essential oil were 100% efficient on the larval stage. In addition, in the Larval Package Test, a mortality of 98.37% was obtained with TpEtOH (LC50 = 3.798 mg mL-1). From the microscopic analysis of ovary cells, morphological changes in the chorion and cytoplasm of oocytes were observed, with vacuolization around the germinal vesicle and disorganization of the pedicular cells, suggesting an interference in the normal embryogenic formation of the larvae. These results demonstrate that T. patula extracts interfere at all stages of development of the ixodid, from eggs to adults forms, and have a pronounced repellent effect. In addition, the results of the cytotoxicity assays performed on keratinocytes, as well as previous information on oral and dermal acute toxicity (LD50 > 4000 mg kg-1), attest that T. patula can be a safe alternative for ectoparasitic control, representing an alternative for development of a formulation used to help control populations of ticks in the field.

Ecological Insights to Track Cytotoxic Compounds among Maytenus ilicifolia Living Individuals and Clones of an Ex Situ Collection.

Biodiversity is key for maintenance of life and source of richness. Nevertheless, concepts such as phenotype expression are also pivotal to understand how chemical diversity varies in a living organism. Sesquiterpene pyridine alkaloids (SPAs) and quinonemethide triterpenes (QMTs) accumulate in root bark of Celastraceae plants. However, despite their known bioactive traits, there is still a lack of evidence regarding their ecological functions. Our present contribution combines analytical tools to study clones and individuals of Maytenus ilicifolia (Celastraceae) kept alive in an ex situ collection and determine whether or not these two major biosynthetic pathways could be switched on simultaneously. The relative concentration of the QMTs maytenin (1) and pristimerin (2), and the SPA aquifoliunin E1 (3) were tracked in raw extracts by HPLC-DAD and ¹H-NMR. Hierarchical Clustering Analysis (HCA) was used to group individuals according their ability to accumulate these metabolites. Semi-quantitative analysis showed an extensive occurrence of QMT in most individuals, whereas SPA was only detected in minor abundance in five samples. Contrary to QMTs, SPAs did not accumulate extensively, contradicting the hypothesis of two different biosynthetic pathways operating simultaneously. Moreover, the production of QMT varied significantly among samples of the same ex situ collection, suggesting that the terpene contents in root bark extracts were not dependent on abiotic effects. HCA results showed that QMT occurrence was high regardless of the plant age. This data disproves the hypothesis that QMT biosynthesis was age-dependent. Furthermore, clustering analysis did not group clones nor same-age samples together, which might reinforce the hypothesis over gene regulation of the biosynthesis pathways. Indeed, plants from the ex situ collection produced bioactive compounds in a singular manner, which postulates that rhizosphere environment could offer ecological triggers for phenotypical plasticity.

Crude leaf extracts of Piperaceae species downmodulate inflammatory responses by human monocytes.

In this study, we aimed to evaluate the immunomodulatory effects of crude leaf extracts from Piper gaudichaudianum Kunth, P. arboreum Aub., P. umbellata L., P. fuligineum Kunth, and Peperomia obtusifolia A. Dietr. on an in vitro model of inflammatory response. The crude extracts were previously obtained by maceration of the leaves. The half-maximal inhibitory concentration was determined by the MTT assay using human peripheral blood mononuclear cells. Human monocytes were simultaneously challenged with each crude extract and lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, to induce a strong inflammatory response. After 24 h of incubation, cell-free supernatants were used for evaluating the mediators involved in inflammation: H2O2, TNF-α, IL-8, IL-6, IL-1ß, IL-10, IL-12, FGF-b, and TGF-ß1. We also compared the results with the effects of ketoprofen, a well-known anti-inflammatory drug. The P. gaudichaudianum crude extract downmodulated the production of H2O2, IL-1ß, IL-6, IL-8, and TGF-ß1 by LPS-stimulated monocytes; P. arboreum, IL-1ß, IL-6, IL-8, and TNF-α; P. umbellata and P. fuligineum, H2O2, IL-1ß, IL-6, IL-8, IL-10, and TNF-α; and P. obtusifolia, H2O2, IL-6, IL-8, IL-10, and TNF-α. In general, the crude leaf extracts amplified the anti-inflammatory response when compared with ketoprofen, particularly reducing the production of IL-8, a mediator involved in neutrophil recruitment during tissue damage. Thus, the crude leaf extracts of P. gaudichaudianum, P. arboreum, P. umbellata, P. fuligineum, and Peperomia obtusifolia elicited an anti-inflammatory response against LPS-challenged monocytes. These findings show the anti-inflammatory properties of these crude leaf extracts and offer new perspectives for their use in the treatment of inflammatory diseases.

Trypanosoma cruzi: analysis of two different strains after piplartine treatment.

The hemoflagellate protozoan, Trypanosoma cruzi, mainly transmitted by triatomine insects through blood transfusion or from mother-to-child, causes Chagas' disease. This is a serious parasitic disease that occurs in Latin America, with considerable social and economic impact. Nifurtimox and benznidazole, drugs indicated for treating infected persons, are effective in the acute phase, but poorly effective during the chronic phase. Therefore, it is extremely urgent to find innovative chemotherapeutic agents and/or effective vaccines. Since piplartine has several biological activities, including trypanocidal activity, the present study aimed to evaluate it on two T. cruzi strains proteome. Considerable changes in the expression of some important enzymes involved in parasite protection against oxidative stress, such as tryparedoxin peroxidase (TXNPx) and methionine sulfoxide reductase (MSR) was observed in both strains. These findings suggest that blocking the expression of the two enzymes could be potential targets for therapeutic studies.

Trypanosoma cruzi: analysis of two different strains after piplartine treatment

ABSTRACT The hemoflagellate protozoan, Trypanosoma cruzi, mainly transmitted by triatomine insects through blood transfusion or from mother-to-child, causes Chagas' disease. This is a serious parasitic disease that occurs in Latin America, with considerable social and economic impact. Nifurtimox and benznidazole, drugs indicated for treating infected persons, are effective in the acute phase, but poorly effective during the chronic phase. Therefore, it is extremely urgent to find innovative chemotherapeutic agents and/or effective vaccines. Since piplartine has several biological activities, including trypanocidal activity, the present study aimed to evaluate it on two T. cruzi strains proteome. Considerable changes in the expression of some important enzymes involved in parasite protection against oxidative stress, such as tryparedoxin peroxidase (TXNPx) and methionine sulfoxide reductase (MSR) was observed in both strains. These findings suggest that blocking the expression of the two enzymes could be potential targets for therapeutic studies.

Screening of 2A peptides for polycistronic gene expression in yeast.

A complexity of pathway expression in yeast compared to prokaryotes is the need for separate promoters and terminators for each gene expressed. Single transcript expression and separated protein production is possible via the use of 2A viral peptides, but detailed characterization to assess their suitability and applications is needed. The present work aimed to characterize multiple 2A peptide sequences to determine suitability for metabolic engineering applications in Saccharomyces cerevisiae. We screened 22 peptides placed between fluorescent protein sequences. Cleaving efficiency was calculated by western blot intensity of bands corresponding to the cleaved and uncleaved forms of the reporter. Three out of the 22 sequences showed high cleavage efficiency: 2A peptide from Equine rhinitis B virus (91%), Porcine teschovirus-1 (85%) and Operophtera brumata cypovirus-18 (83%). Furthermore, expression of the released protein was comparable to its monocistronic expression. As a proof-of-concept, the triterpene friedelin was successfully produced in the same yeast strain by expressing its synthase with the truncated form of HMG1 linked by the 2A peptide of ERBV-1, with production titers comparable to monocistronic expression (via separate promoters). These results suggest that these peptides could be suitable for expression and translation of multiple proteins in metabolic engineering applications in S. cerevisiae.

Friedelin in Maytenus ilicifolia Is Produced by Friedelin Synthase Isoforms.

Triterpenes are interesting compounds because they play an important role in cell homeostasis and a wide variety exhibiting defense functions is produced by plant secondary metabolism. Those same plant secondary metabolites also exhibit biological properties with promising therapeutic potential as anti-inflammatory and antitumor agents. Friedelin is a triterpene ketone with anti-inflammatory and gastroprotective activities and it is a precursor of relevant antitumor quinonemethides. Although many triterpene synthases have been described, only two friedelin synthases were characterized and there is no information about their genomic features and alleles. In the present work, we aimed to identify the gene and new isoforms of friedelin synthase in Maytenus ilicifolia leaves to be functionally characterized in Saccharomyces cerevisiae. The gene sequence analysis elucidated the exon/intron structure and confirmed the presence of single nucleotide polymorphisms with four non-synonymous mutations outside the active site of the enzyme. Therefore, two new isoforms were observed and the heterologous production of the enzymes in yeast showed similar production of friedelin. This first description of different alleles of the gene of friedelin synthase in M. ilicifolia can guide their validation as markers for friedelin-producer specimens.

Piperlongumine and some of its analogs inhibit selectively the human immunoproteasome over the constitutive proteasome.

The natural small molecule piperlongumine A is toxic selectively to cancer cells in vitro and in vivo. This toxicity has been correlated with cancer cell ROS, DNA damage and apoptotic cell death increases. We demonstrate here a new mechanistic property of piperlongumine: it inhibits selectively human immunoproteasome with no noticeable inhibition of human constitutive proteasome. This result suggests that immunoproteasome inhibition, a mechanism independent of ROS elevation, may also partly play a role in the anticancer effects observed with piperlongumine. Structure-activity relationships of piperlongumine analogs suggest that the lactam (piperidonic) ring of piperlongumine A may be replaced by the linear olefin -NHCO-CH2=CH2 to improve both in vitro inhibitory efficiency against immunoproteasome and cellular toxicity.

Chemical Composition and In vitro Anthelmintic Activity of Extracts of Tagetes patula Against a Multidrug-Resistant Isolate of Haemonchus contortus.

Sheep breeding has suffered economic losses due to parasitism by gastrointestinal nematodes, particularly Haemonchus contortus. The use of natural products, specifically Tagetes patula, has been suggested as an alternative method of combatting this issue. Chemical analyses of the extracts of this species described in the literature report the presence of important classes of secondary metabolites such as thiophenes, flavonoids, alkaloids, and benzofurans, some of which were identified and isolated in this study. The aim of this work was to test the effect of the essential oil (EO) and the ethanolic extract of the aerial parts (TpEtOH ) of T. patula on eggs and larvae of H. contortus, through an egg hatch test (EHT) and a larval development test (LDT). In the EHT, the EO showed 100% inhibition at 0.75 mg mL-1 (LC50 = 0.0780 mg mL-1 ), and the TpEtOH showed 100% inhibition at 100 mg mL-1 (LC50 = 12.8 mg mL-1 ). In the LDT, the EO showed 100% inhibition at 0.375 mg mL-1 (LC50 = 0.0400 mg mL-1 ), and the TpEtOH showed 100% inhibition at 1.56 mg mL-1 (LC50 = 0.340 mg mL-1 ). Compared to available literature data, the results presented here suggest that the crude extracts of T. patula have substantial potential for controlling this nematode by interrupting its life cycle and/or preventing it from reaching the infective stage.

The Combined Use of Proteomics and Transcriptomics Reveals a Complex Secondary Metabolite Network in Peperomia obtusifolia.

Peperomia obtusifolia, an ornamental plant from the Piperaceae family, accumulates a series of secondary metabolites with interesting biological properties. From a biosynthesis standpoint, this species produces several benzopyrans derived from orsellinic acid, which is a polyketide typically found in fungi. Additionally, the chiral benzopyrans were reported as racemic and/or as diastereomeric mixtures, which raises questions about the level of enzymatic control in the cyclization step for the formation of the 3,4-dihydro-2H-pyran moiety. Therefore, this article describes the use of shotgun proteomic and transcriptome studies as well as phytochemical profiling for the characterization of the main biosynthesis pathways active in P. obtusifolia. This combined approach resulted in the identification of a series of proteins involved in its secondary metabolism, including tocopherol cyclase and prenyltransferases. The activity of these enzymes was supported by the phytochemical profiling performed in different organs of P. obtusifolia. However, the polyketide synthases possibly involved in the production of orsellinic acid could not be identified, suggesting that orsellinic acid may be produced by endophytes intimately associated with the plant.