Treating leishmaniasis in Amazonia, part 2: Multi-target evaluation of widely used plants to understand medicinal practices.

ETHNOPHARMACOLOGICAL RELEVANCE: Leishmaniasis are widely distributed among tropical and subtropical countries, and remains a crucial health issue in Amazonia. Indigenous groups across Amazonia have developed abundant knowledge about medicinal plants related to this pathology. AIM OF THE STUDY: We intent to explore the weight of different pharmacological activities driving taxa selection for medicinal use in Amazonian communities. Our hypothesis is that specific activity against Leishmania parasites is only one factor along other (anti-inflammatory, wound healing, immunomodulating, antimicrobial) activities. MATERIALS AND METHODS: The twelve most widespread plant species used against leishmaniasis in Amazonia, according to their cultural and biogeographical importance determined through a wide bibliographical survey (475 use reports), were selected for this study. Plant extracts were prepared to mimic their traditional preparations. Antiparasitic activity was evaluated against promastigotes of reference and clinical New-World strains of Leishmania (L. guyanensis, L. braziliensis and L. amazonensis) and L. amazonensis intracellular amastigotes. We concurrently assessed the extracts immunomodulatory properties on PHA-stimulated human PBMCs and RAW264.7 cells, and on L. guyanensis antigens-stimulated PBMCs obtained from Leishmania-infected patients, as well as antifungal activity and wound healing properties (human keratinocyte migration assay) of the selected extracts. The cytotoxicity of the extracts against various cell lines (HFF1, THP-1, HepG2, PBMCs, RAW264.7 and HaCaT cells) was also considered. The biological activity pattern of the extracts was represented through PCA analysis, and a correlation matrix was calculated. RESULTS: Spondias mombin L. bark and Anacardium occidentale L. stem and leaves extracts displayed high anti-promatigotes activity, with IC50 ≤ 32 µg/mL against L. guyanensis promastigotes for S. mombin and IC50 of 67 and 47 µg/mL against L. braziliensis and L. guyanensis promastigotes, respectively, for A. occidentale. In addition to the antiparasitic effect, antifungal activity measured against C. albicans and T. rubrum (MIC in the 16-64 µg/mL range) was observed. However, in the case of Leishmania amastigotes, the most active species were Bixa orellana L. (seeds), Chelonantus alatus (Aubl.) Pulle (leaves), Jacaranda copaia (Aubl.) D. Don. (leaves) and Plantago major L. (leaves) with IC50 < 20 µg/mL and infection rates of 14-25% compared to the control. Concerning immunomodulatory activity, P. major and B. orellana were highlighted as the most potent species for the wider range of cytokines in all tested conditions despite overall contrasting results depending on the model. Most of the species led to moderate to low cytotoxic extracts except for C. alatus, which exhibited strong cytotoxic activity in almost all models. None of the tested extracts displayed wound healing properties. CONCLUSIONS: We highlighted pharmacologically active extracts either on the parasite or on associated pathophysiological aspects, thus supporting the hypothesis that antiparasitic activities are not the only biological factor useful for antileishmanial evaluation. This result should however be supplemented by in vivo studies, and attracts once again the attention on the importance of the choice of biological models for an ethnophamacologically consistent study. Moreover, plant cultural importance, ecological status and availability were discussed in relation with biological results, thus contributing to link ethnobotany, medical anthropology and biology.

Antiparasitic Ovalicin Derivatives from Pseudallescheria boydii, a Mutualistic Fungus of French Guiana Termites.

Social insects are in mutualism with microorganisms, contributing to their resistance against infectious diseases. The fungus Pseudallescheria boydii SNB-CN85 isolated from termites produces ovalicin derivatives resulting from the esterification of the less hindered site of the ovalicin epoxide by long-chain fatty acids. Their structures were elucidated using spectroscopic analysis and semisynthesis from ovalicin. For ovalicin, these compounds displayed antiprotozoal activities against Plasmodium falciparum and Trypanosoma brucei, with IC50 values of 19.8 and 1.1 µM, respectively, for the most active compound, i.e., ovalicin linoleate. In parallel, metabolomic profiling of a collection of P. boydii strains associated with termites made it possible to highlight this class of compounds together with tyroscherin derivatives in all strains. Finally, the complete genome of P. boydii strains was obtained by sequencing, and the cluster of potential ovalicin and ovalicin biosynthesis genes was annotated. Through these metabolomic and genomic analyses, a new ovalicin derivative named boyden C, in which the 6-membered ring of ovalicin was opened by oxidative cleavage, was isolated and structurally characterized.

Identification and dereplication of endophytic Colletotrichum strains by MALDI TOF mass spectrometry and molecular networking.

The chemical diversity of biologically active fungal strains from 42 Colletotrichum, isolated from leaves of the tropical palm species Astrocaryum sciophilum collected in pristine forests of French Guiana, was investigated. The collection was first classified based on protein fingerprints acquired by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) correlated with cytotoxicity. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) data from ethyl acetate extracts were acquired and processed to generate a massive molecular network (MN) using the MetGem software. From five Colletotrichum strains producing cytotoxic specialized metabolites, we predicted the occurrence of peptide and cytochalasin analogues in four of them by MN, including a similar ion clusters in the MN algorithm provided by MetGem software. Chemoinformatics predictions were fully confirmed after isolation of three pentacyclopeptides (cyclo(Phe-Leu-Leu-Leu-Val), cyclo(Phe-Leu-Leu-Leu-Leu) and cyclo(Phe-Leu-Leu-Leu-Ile)) and two cytochalasins (cytochalasin C and cytochalasin D) exhibiting cytotoxicity at the micromolar concentration. Finally, the chemical study of the last active cytotoxic strain BSNB-0583 led to the isolation of four colletamides bearing an identical decadienamide chain.

Alsinol, an arylamino alcohol derivative active against Plasmodium, Babesia, Trypanosoma, and Leishmania: past and new outcomes.

Malaria, babesiosis, trypanosomosis, and leishmaniasis are some of the most life-threatening parasites, but the range of drugs to treat them is limited. An effective, safe, and low-cost drug with a large activity spectrum is urgently needed. For this purpose, an aryl amino alcohol derivative called Alsinol was resynthesized, screened in silico, and tested against Plasmodium, Babesia, Trypanosoma, and Leishmania. In silico Alsinol follows the Lipinski and Ghose rules. In vitro it had schizontocidal activity against Plasmodium falciparum and was able to inhibit gametocytogenesis; it was particularly active against late gametocytes. In malaria-infected mice, it showed a dose-dependent activity similar to chloroquine. It demonstrated a similar level of activity to reference compounds against Babesia divergens, and against promastigotes, and amastigotes stages of Leishmania in vitro. It inhibited the in vitro growth of two African animal strains of Trypanosoma but was ineffective in vivo in our experimental conditions. It showed moderate toxicity in J774A1 and Vero cell models. The study demonstrated that Alsinol has a large spectrum of activity and is potentially affordable to produce. Nevertheless, challenges remain in the process of scaling up synthesis, creating a suitable clinical formulation, and determining the safety margin in preclinical models.

Towards the optimization of botanical insecticides research: Aedes aegypti larvicidal natural products in French Guiana.

Natural products have proven to be an immeasurable source of bioactive compounds. The exceptional biodiversity encountered in Amazonia, alongside a rich entomofauna and frequent interactions with various herbivores is the crucible of a promising chemodiversity. This prompted us to search for novel botanical insecticides in French Guiana. As this French overseas department faces severe issues linked to insects, notably the strong incidence of vector-borne infectious diseases, we decided to focus our research on products able to control the mosquito Aedes aegypti. We tested 452 extracts obtained from 85 species originating from 36 botanical families and collected in contrasted environments against an Ae. aegypti laboratory strain susceptible to all insecticides, and a natural population resistant to both pyrethroid and organophosphate insecticides collected in Cayenne for the most active of them. Eight species (Maytenus oblongata Reissek, Celastraceae; Costus erythrothyrsus Loes., Costaceae; Humiria balsamifera Aubl., Humiriaceae; Sextonia rubra (Mez) van der Werff, Lauraceae; Piper hispidum Sw., Piperaceae; Laetia procera (Poepp.) Eichl., Salicaceae; Matayba arborescens (Aubl.) Radlk., Sapindaceae; and Cupania scrobitulata Rich., Sapindaceae) led to extracts exhibiting more than 50% larval mortality after 48 h of exposition at 100 µg/mL against the natural population and were considered active. Selectivity and phytochemistry of these extracts were therefore investigated and discussed, and some active compounds highlighted. Multivariate analysis highlighted that solvents, plant tissues, plant family and location had a significant effect on mortality while light, available resources and vegetation type did not. Through this case study we highlighted that plant defensive chemistry mechanisms are crucial while searching for novel insecticidal products.

Ilicicolinic acids and ilicicolinal derivatives from the fungus Neonectria discophora SNB-CN63 isolated from the nest of the termite Nasutitermes corniger found in French Guiana show antimicrobial activity.

The fungus Neonectria discophora SNB-CN63 has been isolated from the nest of the termite Nasutitermes corniger found in French Guiana. From the ethyl acetate extract of fungal culture, bioassay guided fractionation led to the isolation of fourteen ilicicolinic acids and ilicicolinal derivatives. Their structures were elucidated by analyses of 1D and 2D NMR and MS spectroscopic data. All metabolites were tested against several microbial pathogens and six displayed antimicrobial activities with MIC <16 µg/mL on T. rubrum, Staphylococcus aureus and methicillin-resistant S. aureus. Based on their structural similarities, a common biosynthetic pathway is proposed for all isolated metabolites.

Aedes aegypti Larvicidal Sesquiterpene Alkaloids from Maytenus oblongata.

Four new sesquiterpene alkaloids (1-4) with a ß-dihydroagrofuran skeleton and a new triterpenoid (5) were isolated from an ethyl acetate extract of Maytenus oblongata stems. Their structures were elucidated using 1D and 2D NMR spectroscopy as well as MS and ECD experiments. The M. oblongata stem EtOAc extract and the pure compounds isolated were tested for larvicidal activity against Aedes aegypti under laboratory conditions, and compounds 2 and 3 were found to be active.

Wayanin and guaijaverin, two active metabolites found in a Psidium acutangulum Mart. ex DC (syn. P. persoonii McVaugh) (Myrtaceae) antimalarial decoction from the Wayana Amerindians.

ETHNOPHARMACOLOGICAL RELEVANCE: Psidium acutangulum Mart. ex DC is a small tree used by the Wayana Amerindians from the Upper-Maroni in French Guiana for the treatment of malaria. AIM OF THE STUDY: In a previous study, we highlighted the in vitro antiplasmodial, antioxidant and anti-inflammatory potential of the traditional decoction of P. acutangulum aerial parts. Our goal was then to investigate on the origin of the biological activity of the traditional remedy, and eventually characterize active constituents. MATERIALS AND METHODS: Liquid-liquid extractions were performed on the decoction, and the antiplasmodial activity evaluated against chloroquine-resistant FcB1 ([(3)H]-hypoxanthine bioassay) and 7G8 (pLDH bioassay) P. falciparum strains, and on a chloroquine sensitive NF54 ([(3)H]-hypoxanthine bioassay) P. falciparum strain. The ethyl acetate fraction (D) was active and underwent bioguided fractionation. All the isolated compounds were tested on P. falciparum FcB1 strain. In vitro anti-inflammatory activity (IL-1ß, IL-6, IL-8, TNFα) of the ethyl acetate fraction and of an anti-Plasmodium active compound, was concurrently assessed on LPS-stimulated human PBMC and NO secretion inhibition was measured on LPS stimulated RAW murine macrophages. Cytotoxicity of the fractions and pure compounds was measured on VERO cells, L6 mammalian cells, PBMCs, and RAW cells. RESULTS: Fractionation of the ethyl acetate soluble fraction (IC50 ranging from 3.4 to <1µg/mL depending on the parasite strain) led to the isolation of six pure compounds: catechin and five glycosylated quercetin derivatives. These compounds have never been isolated from this plant species. Two of these compounds (wayanin and guaijaverin) were found to be moderately active against P. falciparum FcB1 in vitro (IC50 5.5 and 6.9µM respectively). We proposed the name wayanin during public meetings organized in June 2015 in the Upper-Maroni villages, in homage to the medicinal knowledge of the Wayana population. At 50µg/mL, the ethyl acetate fraction (D) significantly inhibited IL-1ß secretion (-46%) and NO production (-21%), as previously observed for the decoction. The effects of D and guiajaverin (4) on the secretion of other cytokines or NO production were not significant. CONCLUSIONS: The confirmed antiplasmodial activity of the ethyl acetate soluble fraction of the decoction and of the isolated compounds support the previous results obtained on the P. acutangulum decoction. The antiplasmodial activity might be due to a mixture of moderately active non-toxic flavonoids. The anti-inflammatory activities were less marked for ethyl acetate fraction (D) than for the decoction.

Therapeutic switching: from antidermatophytic essential oils to new leishmanicidal products.

This study examined whether the antidermatophytic activity of essential oils (EOs) can be used as an indicator for the discovery of active natural products against Leishmania amazonensis. The aerial parts of seven plants were hydrodistilled. Using broth microdilution techniques, the obtained EOs were tested against three strains of dermatophytes (Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis). To compare the EOs antifungal and antiparasitic effects, the EOs activities against axenic amastigotes of L. amazonensis were concurrently evaluated. For the most promising EOs, their antileishmanial activities against parasites infecting peritoneal macrophages of BALB/c mice were measured. The most interesting antifungal candidates were the EOs from Cymbopogon citratus, Otacanthus azureus and Protium heptaphyllum, whereas O. azureus, Piper hispidum and P. heptaphyllum EOs exhibited the lowest 50% inhibitory concentration (IC50) values against axenic amastigotes, thus revealing a certain correspondence between both activities. The P. hispidum EO was identified as the most promising product in the results from the infected macrophages model (IC50: 4.7 µg/mL, safety index: 8). The most abundant compounds found in this EO were sesquiterpenes, notably curzerene and furanodiene. Eventually, the evaluation of the antidermatophytic activity of EOs appears to be an efficient method for identifying new potential drugs for the treatment of L. amazonensis.

Antiplasmodial and anti-inflammatory effects of an antimalarial remedy from the Wayana Amerindians, French Guiana: takamalaimë (Psidium acutangulum Mart. ex DC., Myrtaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Field investigations highlighted the use of Psidium acutangulum Mart. ex DC (syn. P. persoonii McVaugh), a small tree used by the Wayana Amerindians in Twenke-Taluhwen and Antecume-Pata, French Guiana, for the treatment of malaria, and administered either orally in the form of a decoction or applied externally over the whole body. This use appears limited to the Wayana cultural group in French Guiana and has never been reported anywhere else. Our goal was to evaluate the antimalarial and anti-inflammatory activities of a P. acutangulum decoction to explain the good reputation of this remedy. MATERIALS AND METHODS: Interviews with the Wayana inhabitants of Twenke-Taluhwen and Antecume-Pata were conducted within the TRAMAZ project according to the TRAMIL methodology, which is based on a quantitative and qualitative analysis of medicinal plant uses. A decoction of dried aerial parts of P. acutangulum was prepared in consistency with the Wayana recipe. In vitro antiplasmodial assays were performed on chloroquine-resistant FcB1 ([(3)H]-hypoxanthine bioassay) and 7G8 (pLDH bioassay) P. falciparum strains and on chloroquine sensitive NF54 ([(3)H]-hypoxanthine bioassay) P. falciparum strain. In vitro anti-inflammatory activity (IL-1ß, IL-6, IL-8, TNFα) was evaluated on LPS-stimulated human PBMC and NO secretion inhibition was measured on LPS stimulated RAW murine macrophages. Cytotoxicity of the decoction was measured on L6 mammalian cells, PBMCs, and RAW cells. A preliminary evaluation of the in vivo antimalarial activity of the decoction, administered orally twice daily, was assessed by the classical four-day suppressive test against P. berghei NK65 in mice. RESULTS: The decoction displayed a good antiplasmodial activity in vitro against the three tested strains, regardless to the bioassay used, with IC50 values of 3.3µg/mL and 10.3µg/mL against P. falciparum FcB1 and NF54, respectively and 19.0µg/mL against P. falciparum 7G8. It also exhibited significant anti-inflammatory activity in vitro in a dose dependent manner. At a concentration of 50µg/mL, the decoction inhibited the secretion of the following pro-inflammatory cytokines: TNFα (-18%), IL-1ß (-58%), IL-6 (-32%), IL-8 (-21%). It also exhibited a mild NO secretion inhibition (-13%) at the same concentration. The decoction was non-cytotoxic against L6 cells (IC50>100µg/mL), RAW cells and PBMC. In vivo, 150µL of the decoction given orally twice a day (equivalent to 350mg/kg/day of dried extract) inhibited 39.7% average parasite growth, with more than 50% of inhibition in three mice over five. The absence of response for the two remaining mice, however, induced a strong standard deviation. CONCLUSIONS: This study highlighted the in vitro antiplasmodial activity of the decoction of P. acutangulum aerial parts, used by Wayana Amerindians from the Upper-Maroni in French Guiana in case of malaria. Its antioxidant and anti-inflammatory potential, which may help to explain its use against this disease, was demonstrated using models of artificially stimulated cells.

Therapeutic switching: from antidermatophytic essential oils to new leishmanicidal products

This study examined whether the antidermatophytic activity of essential oils (EOs) can be used as an indicator for the discovery of active natural products against Leishmania amazonensis. The aerial parts of seven plants were hydrodistilled. Using broth microdilution techniques, the obtained EOs were tested against three strains of dermatophytes (Trichophyton mentagrophytes, Microsporum gypseum and Microsporum canis). To compare the EOs antifungal and antiparasitic effects, the EOs activities against axenic amastigotes of L. amazonensis were concurrently evaluated. For the most promising EOs, their antileishmanial activities against parasites infecting peritoneal macrophages of BALB/c mice were measured. The most interesting antifungal candidates were the EOs from Cymbopogon citratus, Otacanthus azureus and Protium heptaphyllum, whereas O. azureus, Piper hispidum and P. heptaphyllum EOs exhibited the lowest 50% inhibitory concentration (IC50) values against axenic amastigotes, thus revealing a certain correspondence between both activities. The P. hispidum EO was identified as the most promising product in the results from the infected macrophages model (IC50: 4.7 µg/mL, safety index: 8). The most abundant compounds found in this EO were sesquiterpenes, notably curzerene and furanodiene. Eventually, the evaluation of the antidermatophytic activity of EOs appears to be an efficient method for identifying new potential drugs for the treatment of L. amazonensis.

Antibacterial ilicicolinic acids C and D and ilicicolinal from Neonectria discophora SNB-CN63 isolated from a termite nest.

Ilicicolinic acids A, C, and D (1-3) and ilicicolinal (4) were isolated from a fungus isolated from a Nasutitermes corniger nest in French Guiana. The structures of ilicicolinic acids C and D and ilicicolinal were elucidated using 1D and 2D NMR spectroscopic data as well as MS data. Ilicicolinic acids show antibacterial activity in vitro.

Antimicrobial and cytotoxic secondary metabolites from tropical leaf endophytes: Isolation of antibacterial agent pyrrocidine C from Lewia infectoria SNB-GTC2402.

Because of the symbiotic nature of endophytes, this survey aims to investigate the probability of discovering antibacterial, antifungal and cytotoxic activities in leaf endophytic microbes. We isolated 138 cultivable microbes (121 fungi, 3 bacteria and 14 unidentified or unknown microbes) from 24 plant species, a significant relative proportion of which exhibited antifungal and cytotoxic potential against Candida albicans ATCC 10213 and the human cell lines KB (uterine cervical carcinoma), MDA-MB-435 (melanoma), and MRC5 (normal human lung fibroblasts). Three active fungal extracts were fractionated, resulting in the isolation of eight compounds. Seven had been described in the literature including the following: acremonisol A, semicochliodinol A, cochliodinol, griseofulvin, pyrenocin A, novae zelandin A and alterperylenol. A previously unreported compound named pyrrocidine C was isolated from Lewia infectoria SNB-GTC2402 and identified by spectroscopic analysis. As in pyrrocidines A and B, this compound is a cis-substituted decahydrofluorene with a quaternary carbon at C-5 and opposite stereochemistry at C-8 corresponding to C-6 of pyrrocidines A and B.

Larvicidal activity of isoflavonoids from Muellera frutescens extracts against Aedes aegypti.

The biological activity of extracts from the leaves, bark and roots of Muellera frutescens, an Amazonian ichtyotoxic plant, were evaluated to find new environmentally safe insecticides. The n-hexane extracts of bark, leaf, and root showed a strong toxic activity against Aedes aegypti mosquito larvae. Bioguided fractionation of the bark extract led to the isolation of seven isoflavonoids (12a-hydroxyelliptone, elliptone, (-)-variabilin, rotenone, rotenolone, tephrosin and deguelin). Rotenone and deguelin are responsible for the larvicidal activity of the plant. M frutescens leaves contain up to 0.6%, w/w, deguelin. These results justify the traditional ichtyotoxic use of M frutescens. The leaves contain a relatively high proportion of deguelin and, therefore, can be considered as a renewable source of this environmentally friendly insecticidal isoflavonoid.

The termiticidal activity of Sextonia rubra (Mez) van der Werff (Lauraceae) extract and its active constituent rubrynolide.

BACKGROUND: Termites are degradation agents that inflict severe damage on wood. Some long-lasting Amazonian trees can resist these insects by producing toxic secondary metabolites. These metabolites could potentially replace synthetic termiticidal products which are becoming more restricted to use. RESULTS: Sextonia rubra is resistant to termite-induced degradation. It has been demonstrated that this species naturally produces an ethyl-acetate-soluble termiticidal metabolite, rubrynolide, to protect its wood. Assays in the presence of tropical and invasive termites established that both rubrynolide and crude ethyl acetate extract from S. rubra wood can be used as a treatment for the protection of sensitive woods against termites. CONCLUSION: Rubrynolide and S. rubra extract are promising candidates for the replacement of synthetic termiticides.

Search for antifungal compounds from the wood of durable tropical trees.

Research on antifungal compounds from the durable wood from French Guiana Amazonian forest trees highlights the correlation between the activity of their extracts against wood-rotting fungi and human pathogens. The fractionation of an ethyl acetate extract of Sextonia rubra wood led to the isolation of rubrenolide (1) and rubrynolide (2). The potential of compounds 1 and 2 is described through the evaluation of their activity against 16 pathogenic fungi and their cytotoxicity toward NIH-3T3 mammalian fibroblast cells.

Secondary metabolites of Bagassa guianensis Aubl. wood: a study of the chemotaxonomy of the Moraceae family.

In order to explain the durability of the Moraceae plant family, phytochemistry of Bagassa guianensis was performed. Ethyl acetate extract was obtained from the heartwood and 18 secondary metabolites were isolated, including 6 moracins [6-O-methyl-moracin M, 6-O-methyl-moracin N and moracin Z; previously identified: moracin M, moracin N and moracin P], 8 stilbenoids [presently identified: (-)-epialboctalol and arachidin 4; previously identified: alboctalol, trans-resveratrol, arachidin 2, trans-oxyresveratrol and artogomezianol], 3 previously identified flavonoids, steppogenin, katuranin and dihydromorin, beta-sitosterol and resorcinol. Previous studies suggest that stilbenoids are responsible for the natural durability of wood. Our study has determined that B. guianensis is closely related to Morus sp. in phylogeny and should be included in the Moreae sensu stricto tribe of the Moraceae family.

Antiplasmodial benzophenone derivatives from the root barks of Symphonia globulifera (Clusiaceae).

In an effort to find antimalarial drugs, a systematic in vitro evaluation on a chloroquine-resistant strain of Plasmodium falciparum (FcB1) was undertaken on sixty plant extracts collected in French Guiana. The ethyl acetate extract obtained from the root barks of Symphonia globulifera exhibited a strong antiplasmodial activity (97% at 10 microg/ml). The phytochemical investigation of this extract led to the isolation of nine polycyclic polyprenylated acylphloroglucinol (PPAPs) compounds and two oxidized derivatives. All compounds showed antiplasmodial activity with IC(50)s ranged from 2.1 to 10.1 microM. A LC/ESI-MS(n) study performed on polyprenylated benzophenones previously isolated from Moronobea coccinea provided a reliable method for their detection in the extract and structural elucidation.