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.

Antimicrobial Activity and Synergy Investigation of Hypericum scabrum Essential Oil with Antifungal Drugs.

The chemical composition of Lebanese Hypericum scabrum essential oil (EO) was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GG-MS). Its antimicrobial activity was evaluated by determining its minimal inhibitory concentrations (MICs) against a Gram-negative and a Gram-positive bacterium, one yeast, and five dermatophytes. H. scabrum EO was most active on filamentous fungi (MIC values of 32-64 µg/mL). Synergy within the oil was investigated by testing each of the following major components on Trichophyton rubrum: α-pinene, limonene, myrcene, ß-pinene and nonane, as well as a reconstructed EO. The antifungal activity of the natural oil could not be reached, meaning that its activity might be due, in part, to minor constituent(s). The interactions between H. scabrum EO and commercially available antifungals were assessed by the checkerboard test. A synergistic effect was revealed in the combination of the EO with amphotericin B.

Isolation and Identification of Isocoumarin Derivatives With Specific Inhibitory Activity Against Wnt Pathway and Metabolome Characterization of Lasiodiplodia venezuelensis.

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancers are dependent on Wnt pathway overactivation mostly through dysregulation of pathway component protein expression, which necessitates the search for therapeutically relevant compounds targeting them. Highly diverse microorganisms as endophytes represent an underexplored field in the therapeutic natural products research. In the present work, the objective was to explore the chemical diversity and presence of selective Wnt inhibitors within a unique collection of fungi isolated as foliar endophytes from the long-lived tropical palm Astrocaryum sciophilum. The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation based on its selective activity. Application of geometric transfer from analytical HPLC conditions to semi-preparative scale and use of dry load sample introduction enabled the isolation of 15 pure compounds in a single step. Among the molecules identified, five are original natural products described for the first time, and six are new to this species. An active fraction obtained by semi-preparative HPLC was re-purified by UHPLC-PDA using a 1.7 µm phenyl column. 75 injections of 8 µg were necessary to obtain sufficient amounts of each compound for structure elucidation and bioassays. Using this original approach, in addition to the two major compounds, a third minor compound identified as (R)-(-)-5-hydroxymellein (18) was obtained, which was found to be responsible for the significant Wnt inhibition activity recorded. Further studies of this compound and its structural analogs showed that only 18 acts in a highly specific manner, with no acute cytotoxicity. This compound is notably selective for upstream components of the Wnt pathway and is able to inhibit the proliferation of three triple negative breast cancer cell lines. In addition to the discovery of Wnt inhibitors of interest, this study contributes to better characterize the biosynthetic potential of L. venezuelensis.

Hirtellina lobelii DC. essential oil, its constituents, its combination with antimicrobial drugs and its mode of action.

With the goal of unravelling antimicrobial agents and mixtures inspired by plant defences, we investigated the antibacterial and antifungal efficacy of Hirtellina lobelii DC. essential oil (EO), both alone and in combination with antimicrobial drugs. Hirtellina lobelii DC. EO was analysed by GC, GC-MS and partial fractionation/NMR. It was essentially composed of oxygenated sesquiterpenes (75.2%), with α-bisabolol (34.5%), fokienol (12.0%) and T-muurolol (6.8%) serving as the main components. Microbial susceptibility was determined by the broth microdilution method and was expressed as minimum inhibitory concentration (MIC) and minimum bactericidal or fungicidal concentration (MBC or MFC). This EO was found to possess remarkable bactericidal (MBC/MIC = 2) and fungicidal (MFC/MIC = 1-4) potential, particularly against the Gram (+) bacteria Staphylococcus aureus, including its methicillin-resistant forms, the yeast Cryptococcus neoformans and dermatophytes from the genus Trichophyton (MICs 8-128 µg/ml). The examination of the combined effects of the EO with antimicrobial drugs revealed synergisms of the EO with vancomycin against S. aureus and of the EO with fluconazole and griseofulvin against dermatophytic fungi (FICI 0.2-0.5). The effect of H. lobelii EO on the morphologies of fungal hyphae and bacteria, as determined by scanning electronic microscopy (SEM), showed fungal hyphae swelling and bulging. These results suggest that H. lobelii EO and its major constituent, α-bisabolol, have remarkable antimicrobial potential. Combination therapies of this EO with antifungal drugs could offer a promising alternative for treatment of human mycoses caused by filamentous dermatophytic fungi.

Multiple Streptomyces species with distinct secondary metabolomes have identical 16S rRNA gene sequences.

Microbial diversity studies using small subunit (SSU) rRNA gene sequences continue to advance our understanding of biological and ecological systems. Although a good predictor of overall diversity, using this gene to infer the presence of a species in a sample is more controversial. Here, we present a detailed polyphasic analysis of 10 bacterial strains isolated from three coastal lichens Lichina confinis, Lichina pygmaea and Roccella fuciformis with SSU rRNA gene sequences identical to the type strain of Streptomyces cyaneofuscatus. This analysis included phenotypic, microscopic, genetic and genomic comparisons and showed that despite their identical SSU rRNA sequences the strains had markedly different properties, and could be distinguished as 5 different species. Significantly, secondary metabolites profiles from these strains were also found to be different. It is thus clear that SSU rRNA based operational taxonomy units, even at the most stringent cut-off can represent multiple bacterial species, and that at least for the case of Streptomyces, strain de-replication based on SSU gene sequences prior to screening for bioactive molecules can miss potentially interesting novel molecules produced by this group that is notorious for the production of drug-leads.

Secondary Metabolites Isolated from the Amazonian Endophytic Fungus Diaporthe sp. SNB-GSS10.

We describe a Sabicea cinerea endophytic fungus closely related to Diaporthe pseudomangiferae that produces two known metabolites, mycoepoxydiene (1) and altiloxin A (2), as well as enamidin (3) and eremofortin F (4), two compounds not previously described in the literature. The structure of these four metabolites was elucidated using spectroscopic analysis, and their cytotoxic activities were measured against the human cell lines KB, MRC-5, and MDA-MB-435.

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.

Chemical composition and antimicrobial activity of the essential oil of Ruta chalepensis L. growing wild in Lebanon.

The essential oils (EOs) isolated from the fresh aerial parts of Ruta chalepensis L. collected in North Lebanon were obtained by solvent-free microwave extraction (Milestone®), yielding 0.12% EO from both the leaves and a mixture of stems and leaves. The EOs were characterized by GC/MS analysis, and 27 components were identified, which were primarily ketones (88.0-93.2%). The main components were nonan-2-one and undecan-2-one. The antimicrobial activity of the EOs against a Gram-positive and a Gram-negative bacterium, a yeast, and a dermatophyte was evaluated using the broth-microdilution technique and expressed as minimal inhibitory concentration (MIC). The EOs revealed moderate in vitro antifungal activity against Trichophyton rubrum and Candida albicans.

Chemical composition and antimicrobial activity of the essential oil of Juniperus excelsa M.Bieb. growing wild in Lebanon.

The essential oils (EOs) isolated from the leaves and twigs of Juniperus excelsa M.Bieb. growing wild in Lebanon were characterized, and their antimicrobial activity and antiradical capacity were evaluated. The EOs were obtained by hydrodistillation using a Clevenger-type apparatus and characterized by GC and GC/MS analyses. The antimicrobial activity was evaluated by determining minimal inhibitory concentrations (MICs) against a Gram-positive and a Gram-negative bacterium, a yeast, and a dermatophyte with the broth microdilution technique. A total of 28 constituents was identified and accounted for 90.1 and 95.6% of the twig and leaf EO composition, respectively. Both EOs were essentially composed of monoterpene hydrocarbons (46.7 and 59.6% for twig and leaf EOs, resp.) and sesquiterpenes (39.4 and 32.1%, resp.). The main components were α-pinene, α-cedrol, and δ-car-3-ene. The J. excelsa EOs did not show any antiradical potential, but revealed interesting in vitro antimicrobial activities against Staphylococcus aureus and Trichophyton rubrum (MICs of 64 and 128 µg/ml, resp.). The three major compounds were tested separately and in combination according to their respective amounts in the oil. δ-Car-3-ene was the most active component and is undoubtedly one of the constituents driving the antifungal activity of J. excelsa essential oil, even though synergies are probably involved.

Chemical composition and antinociceptive effect of aqueous extract from Rourea induta Planch. leaves in acute and chronic pain models.

ETHNOPHARMACOLOGICAL RELEVANCE: Rourea induta Planch. is a small tree or shrub growing wild in Brazil and belonging to the Connaraceae family. It is used for the treatment of Chagas disease and as antirheumatic in folk medicine. This study was designed to investigate the antinociceptive activity of the aqueous extract (AERi) of Rourea induta leaves in rodents, as well as isolate and identify components that can be responsible for its effect. MATERIAL AND METHODS: The antinociceptive effect of orally administered AERi was evaluated in behavioral models of acute (formalin) and chronic [complete Freund׳s adjuvant (CFA)] pain in mice. We also investigated the possible involvement of opioid receptors and proinflammatory cytokines (interleukin-1ß and tumor necrosis factor-α) in the antinociceptive effect of the AERi. In addition, a nonspecific effect of AERi was evaluated by measuring locomotor activity and corporal temperature. Finally, we performed a phytochemical analysis of AERi. RESULTS: HPLC titration revealed the presence of hyperin (21.6 mg/g), quercetin-3-O-ß-xyloside (4.4 mg/g), quercetin-3-O-α-arabinofuranoside (12.0 mg/g), and quercetin (2.1 mg/g). It was also possible to isolate minor constituents׳ chlorogenic acid, neochlorogenic acid and procyanidin C1. The oral administration of AERi (100 mg/kg) significantly inhibited the neurogenic (37 ± 5%) and inflammatory (34 ± 7%) phases of formalin-induced pain. Acute and repeated treatment of animals with AERi (100 mg/kg, p.o.) once a day markedly reduced the mechanical hypersensitivity response induced by CFA, and this effect was evident until the day 10. Moreover, repeated treatment with AERi (100 mg/kg, p.o.) significantly reduced the levels of IL-1ß and TNF-α in the paw (22 ± 7% and 50 ± 19%) and in the spinal cord (100% and 100%) when compared to the CFA group. The AERi (100 mg/kg, p.o.) markedly reduced biting behavior induced by intrathecal injection of TNF-α (81 ± 11%). Finally, the effect of AERi was not associated with changes in locomotor activity or the corporal temperature of animals. CONCLUSION: These data show that aqueous extract of Rourea induta has significant antinociceptive action, which seems to be associated with an inhibition of pro-inflammatory cytokines activated pathways. These findings support the ethnomedical uses of this plant.

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.

Antifungal agents from Pseudallescheria boydii SNB-CN73 isolated from a Nasutitermes sp. termite.

Defense mutualisms between social insects and microorganisms have been described in the literature. The present article describes the discovery of a Pseudallescheria boydii strain isolated from Nasutitermes sp. The microbial symbiont produces two antifungal metabolites: tyroscherin and N-methyltyroscherin, a compound not previously described in the literature. Methylation of tyroscherin has confirmed the structure of N-methyltyroscherin. Both compounds are effective antifungal agents with favorable selectivity indices for Candida albicans and Trichophyton rubrum.

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.

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.

Antiplasmodial benzophenones from the trunk latex of Moronobea coccinea (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 methanol extract obtained from the latex of Moronobea coccinea exhibited a strong antiplasmodial activity (95% at 10microg/ml). The phytochemical investigation of this extract led to the isolation of eleven polycyclic polyprenylated acylphloroglucinols (PPAPs), from which eight showed potent antiplasmodial activity with IC50 ranged from 3.3microM to 37.2microM.

Cytotoxic farnesyl glycosides from Pittosporum pancheri.

Bioassay guided purification of the ethanolic extract of the bark of New Caledonian Pittosporum pancheri Brongn. and Gris (Pittosporaceae) led to the isolation and characterization of two new farnesyl monoglycosides, pancherins A and B. The structure of these compounds were determined on the basis of spectroscopic studies. The new compounds displayed a significant activity in the in vitro cytotoxic assay against KB cancer cell line, and pancherin A inhibits weakly farnesyl protein transferase.

Cytotoxic monotetrahydrofuran acetogenins from Disepalum plagioneurum.

The known plagionicin A (1) and eight new monotetrahydrofuran acetogenins, plagionicins B-D (2-4) and plagioneurins A-E (5-9), were isolated from the leaves of Disepalum plagioneurum by bioassay-guided purification. The structures of the new compounds were elucidated by spectroscopic methods. The new monotetrahydrofuran (mono-THF) acetogenins exhibited significant in vitro cytotoxicity against the KB cancer cell line, with IC(50) values in the nanomolar range.