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.

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.

Stryphnodendron Species Known as "Barbatimão": A Comprehensive Report.

Stryphnodendron spp., popularly known as "barbatimão", is the native Brazilian tree most often employed to treat wounds and infections. The aim of the present study was to highlight the importance of S. adstringens, as well as other Stryphnodendron species recognized as "barbatimão", to human health, depicting the relevance of ethnopharmacological knowledge to scientific evidence for uses, related chemical compounds, development of pharmaceutical formulations, and the establishment of toxicity parameters. For this purpose, the literature databases PubMed, Scielo, Lilacs, CAPES Thesis and Google Scholar were searched until 2017. It was observed that stem bark was the primary part of the plant used, mainly as a decoction, for wound healing and treatment of infectious and inflammatory disorders. Confirmed biological activities, including wound healing, anti-inflammatory, antioxidant, and antimicrobial activities, were related to the presence of compounds from tannin class, mostly proanthocyanidins. Toxicity parameters for stem bark were inconclusive, but toxicity was observed to a significant extent when seeds were ingested by cattle or other animals. Due to these important and confirmed biological activities, government policy encourages the phytotherapic use of S. adstringens, and some formulations with stem bark extracts were developed and patented. Furthermore, antiprotozoal, hypoglycemic and antiviral activities were identified as promising.

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.

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.

Anti-Candida targets and cytotoxicity of casuarinin isolated from Plinia cauliflora leaves in a bioactivity-guided study.

In addition to the bio-guided investigation of the antifungal activity of Plinia cauliflora leaves against different Candida species, the major aim of the present study was the search for targets on the fungal cell. The most active antifungal fraction was purified by chromatography and characterized by NMR and mass spectrometry. The antifungal activity was evaluated against five Candida strains according to referenced guidelines. Cytotoxicity against fibroblast cells was determined. The likely targets of Candida albicans cells were assessed through interactions with ergosterol and cell wall composition, porosity and architecture. The chemical major component within the most active antifungal fraction of P. cauliflora leaves identified was the hydrolysable tannin casuarinin. The cytotoxic concentration was higher than the antifungal one. The first indication of plant target on cellular integrity was suggested by the antifungal activity ameliorated when using an osmotic support. The most important target for the tannin fraction studied was suggested by ultrastructural analysis of yeast cell walls revealing a denser mannan outer layer and wall porosity reduced. It is possible to imply that P. cauliflora targeted the C. albicans cell wall inducing some changes in the architecture, notably the outer glycoprotein layer, affecting the cell wall porosity without alteration of the polysaccharide or protein level.

Flavonoids from Plinia cauliflora (Mart.) Kausel (Myrtaceae) with antifungal activity.

Plinia cauliflora is a plant species with edible fruits but its chemical composition is not totally known yet although former studies showed its potential as antifungal agent. This work aimed the chemical analysis of the leaves, the activity against fungi species and evaluated cytotoxicity. Extract was obtained with 70% ethanol. An ethyl acetate fraction was obtained and glycosylated quercetin and myricetin were isolated. Samples were tested against Candida species, dermatophytes and entomopathogenic fungi. Cytotoxicity was evaluated against fibroblast cells. Extract showed good activity against C. albicans (minimum inhibitory concentration at 156 µg/mL), C. parapsilosis (78 µg/mL), C. krusei (19 µg/mL), Trichophyton rubrum (78 µg/mL) and Microsporum canis (156 µg/mL). Isolated compounds were more active against C. krusei and T. rubrum. The extract, which was the more active sample, demonstrated low cytotoxic effect and encourage more studies against rising non-albicans species and dermatophyte T. rubrum.

Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene.

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to ß-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

Phytochemical Characterization, Antimicrobial Activity, and Antioxidant Potential of Equisetum hyemale L. (Equisetaceae) Extracts.

Equisetum hyemale species is considered a medicinal plant used in the form of infusions to combat infectious or inflammation diseases and also diuretic effects, presenting several compounds related to these actions. In previous studies different species of Equisetum showed several phenolic compounds. The objective of this study was, for the first time, based on phytochemistry analysis to evaluate the antioxidant and antimicrobial activity. The 70% ethanolic and methanolic extracts of E. hyemale were characterized by spectrophotometric and high-performance liquid chromatography with pulsed amperometric detector analyses, as well as its antioxidant potential based on the scavenger activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH). In addition was verified the antimicrobial activity by broth microdilution technique against bacteria and fungi. The extracts showed phytochemical similarity, which demonstrated the presence of phenolic compounds, the scavenging activity for free radicals was about 30% and was observed better antifungal activity against dermatophyte fungi, with minimum inhibitory concentration and minimum fungicidal concentration of 0.62 mg/mL to Trichophyton rubrum and Microsporum canis. The extracts exhibits great potential to therapeutic applications or product development, since both possess antifungal activity and antioxidant action associated with little difference in their phytochemical composition.

Antidiarrheal activity of Campomanesia xanthocarpa fruit.

The growing list of drug-resistant microorganisms and the persistence of deaths due to diarrhea are compelling reasons to study plants in search of new therapeutic agents. The chemical constitution and popular use of the edible fruits of Campomanesia xanthocarpa O. Berg motivated this study to assess the antimicrobial and antidiarrheal properties of the fruits. An extract in 70% ethanol was prepared, and its antimicrobial activity was tested against several strains of bacteria by the agar diffusion and microdilution methods. Antidiarrheal activity was analyzed by testing intestinal motility in an animal model. Preliminary phytochemical study indicated the presence of flavonoids, saponins, and tannins in the hydroalcoholic extract. Antimicrobial activity was significant, but the minimum inhibitory concentration proved to be higher than the maximum extract concentration tested. The extract did not show significant activity for intestinal motility. Although this fruit extract did not show great results as an antimicrobial or antidiarrheal agent, the study contributes to the search for new plant agents and could be referred to as a research protocol by investigators in this area.

Chemical and antidiarrheal studies of Plinia cauliflora.

Plinia cauliflora (Mart.) Kausel, widespread in South America, has edible fruits, and its bark is commonly used against diarrhea and other disorders, on account of its astringency. Because diarrhea is still one of the most important causes of illness and death among children in developing countries, where the population turns to traditional medicine for its treatment, the present study determined the composition of fruit and leaf extracts of P. cauliflora, analyzed the activity against diarrhea by antimicrobial and gastrointestinal motility, and evaluated the cytotoxicity of the extracts. Chemical composition was determined by high-performance liquid chromatograpy-ultraviolet/photodiode array detection. Antimicrobial activity was analyzed by agar diffusion and the microdilution method against etiological agents of diarrhea. The effect on gastrointestinal motility was analyzed using an experimental model in mice. Cytotoxicity was evaluated in vitro with the fibroblast cell line SIRC CCL 60, and leaf extract showed a 50% inhibitory concentration of 0.48 µg/mL. Gallic acid, ellagic acid, and flavonoid derivatives were detected in the extracts. It was observed that fruit and leaf extracts showed some activity against Enterococcus faecalis, Escherichia coli, Salmonella sp., and Shigella sp. However, neither extract had any effect on gastrointestinal motility.

O Brasil no contexto de controle de qualidade de plantas medicinais / Brazil in the context of plants and derivates quality control

Atualmente, as plantas medicinais movem altos valores financeiros em todo o mundo e representam o tipo de tratamento mais acessível para cerca de 80 por cento da população, principalmente entre os países em desenvolvimento. Entretanto, existe ainda uma falta de conhecimento sobre propriedades químicas, farmacológicas e toxicológicas a fim de assegurar a eficácia e segurança das plantas medicinais. Os critérios de eficácia e segurança de plantas medicinais estão relacionados a qualidade, isto é, as plantas necessitam ser corretamente identificadas, cultivadas e coletadas, devem estar livres de material estranho, partes de outras plantas e contaminações inorgânicas e/ou microbianas. O objetivo deste estudo foi o de elucidar os diferentes processos e padronizações sobre o controle de drogas vegetais, principalmente no Brasil.

Nowadays medicinal plants move high financial values at the world and represent a type of treatment more accessible for about 80 percent of people, mainly among developing countries. However, there is a lack of knowledge about chemical, pharmacological and toxicological properties towards ensure their efficacy and safety. Efficacy and safety criteria of medicinal plants are related to quality, that is, the plants need to be correctly identified, cultivated and harvested, they must be free of strange material, part of other plants and inorganic and/or microorganisms contamination. The aim of this study was to elucidate different process and standardizations about quality control of vegetal drugs mainly in Brazil.

Histochemical, phytochemical and biological screening of Plinia cauliflora (DC. ) Kausel, Myrtaceae, leaves / Rastreamento histoquímico, fitoquímico e biológico de folhas de Plinia cauliflora (DC. ) Kausel (Myrtaceae)

In this work, chemical and biological activities of crude extracts obtained with 50 percent ethanol, 70 percent ethanol, acetone:water (7:3; v/v) and chloroform of Plinia cauliflora (DC.) Kausel, Myrtaceae, leaves, a native tree from several regions of Brazil, was investigated. Histochemical and phytochemical screenings were done according to characterization reactions and thin layer chromatography. To assist in extracts standardization, total phenol and flavonoids content spectrophotometric was performed. Antioxidant activity was analyzed by percentage of radical scavenging using DPPH solution. Antimicrobial activity was evaluated against Gram-positive, Gram-negative pathogenic bacteria and species of Candida using agar diffusion and minimal inhibitory concentration (MIC) determination methods according to standard methods. The leaves presented lipids at secretory cavity and phenols, mainly tannins, in nervures and palisade parenchyma. Polar extracts showed flavonoids, tannins and high content of phenols and flavonoids. The extracts showed great antioxidant activity and antimicrobial activity was better against Candida species than against bacteria.

No presente trabalho, foram investigadas a composição química e atividades biológicas de extratos brutos obtidos com etanol 50 por cento, etanol 70 por cento, acetona:água (7:3; v/v) e clorofórmio das folhas de Plinia cauliflora (DC.) Kausel, Myrtaceae, uma árvore nativa de várias regiões do Brasil. Os rastreamentos histoquímico e fitoquímico foram feitos de acordo com reações de caracterização e cromatografia em camada delgada. Para auxiliar na padronização dos extratos foram realizadas determinações do teor de fenóis totais e de flavonoides totais, espectrofotometricamente. A atividade antioxidante foi analisada pela porcentagem de sequestro de radicais livres usando solução de DPPH. A atividade antimicrobiana foi avaliada frente a bactérias patogênicas Gram-positivas, Gram-negativas e espécies de Candida utilizando os métodos de difusão em ágar e determinação da concentração inibitória mínima (MIC) de acordo com métodos padronizados. As folhas apresentaram lipídeos nas cavidades secretoras e fenóis, principalmente taninos, nas nervuras e parênquima paliçádico. Os extratos polares apresentaram flavonoides, taninos, alto teor de fenóis totais e de flavonoides totais. Os extratos mostraram elevada atividade antioxidante e a atividade antimicrobiana foi melhor contra as espécies de Candida do que contra as bactérias.