In Vitro and In Silico Evaluation of the Leishmanicidal and Trypanocidal Activities of Lignan Methylpiperitol Isolated from Persea Fulva.

Neglected Tropical Diseases are a significant concern as they encompass various infections caused by pathogens prevalent in tropical regions. The limited and often highly toxic treatment options for these diseases necessitate the exploration of new therapeutic candidates. In the present study, the lignan methylpiperitol was isolated after several chromatographic steps from Persea fulva L. E. Koop (Lauraceae) and its leishmanicidal and trypanocidal activities were evaluated using in vitro and in silico approaches. The chemical structure of methylpiperitol was defined by NMR and MS spectral data analysis. The antiprotozoal activity of methylpiperitol was determined in vitro and indicated potency against trypomastigote forms of Trypanosoma cruzi (EC50 of 4.5±1.1 mM) and amastigote forms of Leishmania infantum (EC50 of 4.1±0.5 mM), with no mammalian cytotoxicity against NCTC cells (CC50>200 mM). Molecular docking studies were conducted using six T. cruzi and four Leishmania. The results indicate that for the molecular target hypoxanthine phosphoribosyl transferase in T. cruzi and piteridine reductase 1 of L. infatum, the methylpiperitol obtained better results than the crystallographic ligand. Therefore, the lignan methylpiperitol, isolated from P. fulva holds potential for the development of new prototypes for the treatment of Neglected Tropical Diseases, especially leishmaniasis.

Evaluation of the anti-inflammatory activity of Acacia polyphylla and identification of a new apigenin-3-C- glycosylated type flavonoid.

Owing to the potentially harmful adverse effects of current anti-inflammatory drugs, there is a need to identify new alternative substances. Thus, this study aimed to perform a phytochemical analysis of A. polyphylla to identify compounds responsible for its anti-inflammatory activity. Several fractions of the A. polyphylla extract were obtained and evaluated in an ex vivo anti-inflammatory assay using fresh human blood. Among the evaluated fractions, the BH fraction displayed the highest percentage of PGE2 inhibition (74.8%) compared to the reference drugs dexamethasone and indomethacin, demonstrating its excellent potential for anti-inflammatory activity. Astragalin (P1), a known 3-O-glucoside of kaempferol, was isolated from the A. polyphylla extract for the first time. In addition, a new compound (P2) was isolated and identified as the apigenin-3-C-glycosylated flavonoid. Astragalin showed moderate PGE2 activity (48.3%), whereas P2 was not anti-inflammatory. This study contributes to the phytochemical studies of A. polyphylla and confirms its anti-inflammatory potential.

Exploring functional and structural features of chemically related natural prenylated hydroquinone and benzoic acid from Piper crassinervium (Piperaceae) on bacterial peroxiredoxin inhibition.

Multiple drug resistance (MDR) bacterial strains are responsible by 1.2 million of human deaths all over the world. The pathogens possess efficient enzymes which are able to mitigate the toxicity of reactive oxygen species (ROS) produced by some antibiotics and the host immune cells. Among them, the bacterial peroxiredoxin alkyl hydroperoxide reductase C (AhpC) is able to decompose efficiently several kinds of hydroperoxides. To decompose their substrates AhpC use a reactive cysteine residue (peroxidatic cysteine-CysP) that together with two other polar residues (Thr/Ser and Arg) comprise the catalytic triad of these enzymes and are involved in the substrate targeting/stabilization to allow a bimolecular nucleophilic substitution (SN2) reaction. Additionally to the high efficiency the AhpC is very abundant in the cells and present virulent properties in some bacterial species. Despite the importance of AhpC in bacteria, few studies aimed at using natural compounds as inhibitors of this class of enzymes. Some natural products were identified as human isoforms, presenting as common characteristics a bulk hydrophobic moiety and an α, ß-unsaturated carbonylic system able to perform a thiol-Michael reaction. In this work, we evaluated two chemically related natural products: 1,4-dihydroxy-2-(3',7'-dimethyl-1'-oxo-2'E,6'-octadienyl) benzene (C1) and 4-hydroxy-2-(3',7'-dimethyl-1'-oxo-2'E,6'-octadienyl) benzoic acid (C2), both were isolated from branches Piper crassinervium (Piperaceae), over the peroxidase activity of AhpC from Pseudomonas aeruginosa (PaAhpC) and Staphylococcus epidermidis (SeAhpC). By biochemical assays we show that although both compounds can perform the Michael addition reaction, only compound C2 was able to inhibit the PaAhpC peroxidase activity but not SeAhpC, presenting IC50 = 20.3 µM. SDS-PAGE analysis revealed that the compound was not able to perform a thiol-Michael addition, suggesting another inhibition behavior. Using computer-assisted simulations, we also show that an acidic group present in the structure of compound C2 may be involved in the stabilization by polar interactions with the Thr and Arg residues from the catalytic triad and several apolar interactions with hydrophobic residues. Finally, C2 was not able to interfere in the peroxidase activity of the isoform Prx2 from humans or even the thiol proteins of the Trx reducing system from Escherichia coli (EcTrx and EcTrxR), indicating specificity for P. aeruginosa AhpC.

Leishmanicidal Activity and Ultrastructural Changes of Maslinic Acid Isolated from Hyptidendron canum.

The therapeutic arsenal for the treatment of leishmaniasis is limited and has serious obstacles, such as variable activity, high toxicity, and costs. To overcome such limitations, it becomes urgent to characterize new bioactive molecules. Plants produce and accumulate different classes of bioactive compounds, and these molecules can be studied as a strategy to combat leishmaniasis. The study presented herein evaluated the leishmanicidal effect of maslinic acid isolated from the leaves of Hyptidendron canum (Lamiaceae) and investigated the morphological that occurred on Leishmania (Leishmania) infantum upon treatment. Maslinic acid was active and selective against promastigote and amastigote forms in a dose-dependent manner. Additionally, it was not toxic to peritoneal macrophages isolated from golden hamsters, while miltefosine and amphotericin B showed mild toxicity for macrophages. Morphological changes in promastigotes of L. (L.) infantum treated with maslinic acid were related to cytoplasmic degeneration, intense exocytic activity, and blebbing in the kDNA; disruption of mitochondrial cristae was observed in some parasites. The nucleus of promastigote forms seems to be degraded and the chromatin fragmented, suggesting that maslinic acid triggers programmed cell death. These results indicate that maslinic acid may be an interesting molecule to develop new classes of drugs against leishmaniasis.

Electrospray mass-spectrometry guided target isolation of neolignans from Nectandra leucantha (Lauraceae) by high performance- and spiral-coil countercurrent chromatography.

Nectandra leucantha (Lauraceae) is a tree indigenous to the tropical Atlantic forests of Brazil, one of the most biodiverse flora hotspots worldwide. This plant species contains high concentrations of neolignan and dehydrodieugenol derivatives that express significant in-vitro activities against various parasite strains. These activities are however responsible for severe tropical human infections, such as Leishmaniasis (Leishmania spp.) and Chagas disease (Trypanosoma cruzi), which have been classified by the World Health Organization (WHO) as Neglected Tropical Diseases (NTDs). In order to optimize the isolation process for these target metabolites, n-hexane extract of the leaves was separated by means of semi-preparative high performance countercurrent chromatography (HPCCC) and scale-up spiral-coil countercurrent chromatography (sp-CCC) systems. Several biphasic solvent mixtures were evaluated for their partitioning effects on neolignans, resulting in the selection of an optimized system n-hexane - ethylacetate - methanol - water (7:3:7:3, v/v/v/v). The chromatographic experiments on the HPCCC and sp-CCC were run in the head-to-tail mode with 500 mg and 16 g injections, respectively. For specific and multiple metabolite detection, the recovered CCC-fractions were off-line injected, in the sequence of recovery, to an electrospray mass-spectrometry (ESI-MS/MS) device. A projection of the single ion traces of the target compounds, in the positive ionization mode at a scan range of m/z 100-1500, located chromatographic areas where the co-elution effects occurred and pure target metabolites were present. Five major target neolignans were specifically detected, which enabled the accurate pooling of CCC-fractions for an optimum recovery of the metabolites. The direct comparison of the performance characteristics of the two CCC-devices, with very different mechanical designs was achieved by the conversion of the time axis into a partition ratio (KD) separation scale. As a result, the compound specific KD-elution values of the target neolignan were determined in high precision, while the comparison of the calculated separation factor (α) and resolution factor (RS) values revealed a superior separation performance for the HPCCC system. Also, the reproducibility of detected metabolites in the two CCC experiments was confirmed by small variations (ΔKD ±0.1). Neolignan target compounds with anti-parasite activities were successfully isolated in the 100 mg to 4 g range in a single lab-scale countercurrent chromatographic process step.

Ethnopharmacology Study of Plants from Atlantic Forest with Leishmanicidal Activity.

Leishmaniasis is an infectious disease caused by a protozoan belonging to Leishmania genus. Different clinical outcomes can be observed depending on the parasite species and patient's health condition. The outcomes can range from single cutaneous lesions to lethal visceral form. The treatment of all forms of leishmaniasis is based on pentavalent antimonials, and, in some cases, the second-line drug, amphotericin B, is used. Beside the toxicity of both classes of drugs, in some areas of the world, parasites are resistant to antimonial. These detrimental features make fundamental the discovery and characterization of new drugs or plant extracts with leishmanicidal effects. Brazil is a well-known country for its biodiversity. Additionally, the common knowledge inherited for generations in small villages makes Brazil a source of new information and resources for the discovery and development of new drugs. Based on ethnopharmacology, elderlies were interviewed about plants they commonly used for skin diseases and infections. Five native plants from Atlantic forest were indicated; EtOH and n-hexane extracts were prepared with the vegetative organs of the plants and assayed against promastigote and amastigote forms of L. (L.) amazonensis. The major molecules of each extract were detected using qualitative nuclear magnetic resonance. Among all tested extracts, the n-hexane extract from the leave of Eugenia uniflora (Myrtaceae), enriched in myricitrin and quercitrin flavonoids, was the most effective against L. (L.) amazonensis amastigotes. This data supports the ethnopharmacology approach as a successful tool for the discovery of new drugs with leishmanicidal effects.

Isolation and characterization of sodium 2-fluoroacetate from Mascagnia rigida using chromatography and infrared spectroscopy.

Sodium monofluoroacetate was first identified in Dichapetalum cymosum, a South African plant that can cause livestock poisoning and death. After, several other plants also showed to contain this toxin, which leads to the "sudden death". Mascagnia rigida, a well identified poisonous plant, commonly found in northeast of Brazil also cause sudden death in cattle, which shows clinical signs similar to those produced by the ingestion of plants that contain monofluoroacetate. Our aim was to identify the toxic compound present in the aqueous extract of M. rigida. For this purpose, the dried and milled plant was extracted; the extract was lyophilized and submitted to successive chromatographic process, until the desired purity of the active compound was achieved. The study of this material by planar chromatography and by infrared spectrometry indicated that the toxin can be a mixture of mono, di and trifluoroacetate.