Billy Cabanillas

Background: The gut microbiota of insects represents an underexplored reservoir of bioactive compounds with potential antimicrobial applications. This study is aimed at identifying antimicrobial compounds from fungi associated with the gut microbiota of Hermetia illucens (black soldier fly) larvae active against Salmonella enterica serovar Pullorum, an important poultry pathogen. Methods: Fungi isolated from H. illucens larval gut were screened for antimicrobial activity against S. enterica serovar Pullorum. The active fungus was identified through molecular analysis of ITS, benA, and CaM regions. Ethyl acetate extract from the fungal culture was assessed for antimicrobial activity, followed by bioguided fractionation using preparative and semipreparative chromatography. Active fractions were analyzed using UHPLC/HRMS, and putative compound identification was performed through mass spectrometry and molecular networking. Result: Aspergillus chevalieri was identified as active against S. enterica serovar Pullorum, with the ethyl acetate extract exhibiting moderate antimicrobial activity (MIC = 4.00 mg/mL; IC ₅₀ = 3.00 ± 0.58 mg/mL). Bioguided fractionation resulted in several active fractions. UHPLC/HRMS analysis putatively annotated 10 compounds, previously shown to be bioactive, including diketopiperazines (Neoechinulin A and echinulin), peptide derivatives (cyclo(L‐Tyr‐L‐Pro) and N ‐acetyltyramine), a benzofuran derivative, an isocoumarin (diaporthin), a flavin (lumichrome), an aminopurine (isopentenyladenine), and two diterpenoids (4‐deoxyphorbol‐13‐acetate and austinoneol). Conclusion: This study represents the first report of A. chevalieri associated with H. illucens larvae and demonstrates its potential as a source of antimicrobial compounds against S. enterica serovar Pullorum. The identified bioactive compounds provide promising leads for the development of new antimicrobial agents for poultry health.

The gut microbiota of insects is composed of a wide range of microorganisms which produce bioactive compounds that protect their host from pathogenic attack. In the present study, we isolate and identify the fungus Chrysosporium multifidum from the gut of Hermetia illucens larvae. Extract from C. multifidum culture broth supernatant showed moderate activity against a strain of methicillin-resistant Staphylococcus aureus (MRSA). Bioguided isolation of the extract resulted in the characterization of six α-pyrone derivatives (1–6) and one diketopiperazine (7). Of these compounds, 5,6-dihydro-4-methoxy-6-(1-oxopentyl)-2H-pyran-2-one (4) showed the greatest activity (IC₅₀ = 11.4 ± 0.7 μg/mL and MIC = 62.5 μg/mL) against MRSA.

The gut microbiota of insects contains a wide range of organisms that protect them against the attack of pathogens by releasing various types of bioactive compounds. In the present study, we report the isolation and identification of the fungus Chrysosporium multifidum as a component of the microbiota from the larval gut of Hermetia illucens. Extract from the broth culture of C. multifidum showed moderate activity on a strain of methicillin-resistant Staphylococcus aureus (MRSA). The bioguided isolation of the extract resulted in the characterization of six α-pyrone derivatives ( 1 - 6 ) and one diketopiperazines ( 7 ), among them 5,6-dihydro-4-methoxy-6-(1-oxopentyl)-2H-pyran-2-one ( 4 ) showed the best activity (IC ₅₀ = 11.4 ± 0.7 µg/ml and MIC = 62.5 μg/ml).

Introduction: The phytochemistry of the latex of Hura crepitans L. (Euphorbiaceae), a widespread tree in the Amazonian forest having many uses, is little known. Only huratoxin, a daphnane diterpene orthoester, has been described despite the high pharmacological potential of this kind of compounds. Glucosphingolipids (cerebrosides) are also known to be distributed in Euphorbiaceae latexes.Objective: To tentatively identify daphnanes diterpenes and cerebrosides in the latex of H. crepitans.Methods: An ethanolic extract of the lyophilised latex of H. crepitans was analysed by ultra‐high‐performance liquid chromatography (UHPLC) coupled with positive and negative atmospheric pressure chemical ionisation high‐resolution mass spectrometry (APCI‐HRMS) method using a quadrupole/linear ion trap/Orbitrap (LTQ‐Orbitrap). Tandem mass spectrometry (MS/MS) spectra were recorded by two different fragmentation modes: collision induced dissociation (CID) and higher‐energy collisional dissociation (HCD).Results: The analysis of CID‐ and HCD‐MS/MS spectra allowed to propose fragmentation patterns for daphnane esters and cerebrosides and highlight diagnostic ions in positive and negative ion modes. A total of 34 compounds including 24 daphnane esters and 10 cerebrosides have been tentatively annotated. Among them, 17 daphnane diterpenes bearing one or two acyl chains are new compounds and the cerebrosides are described in the genus Hura for the first time.Conclusion: This study revealed the chemical constituents of the latex of H. crepitans and particularly its richness and chemical diversity in daphnane diterpenes, more frequently encountered in the species of Thymelaeaceae.

Malaria and leishmaniasis are considered by the WHO as the first and second most important protozoan vector-borne parasitic diseases in the tropics [1]. Traditional medicine in the Peruvian Amazonian regions is commonly used by local people. Forty-eight plants, without previous phytochemical background, were collected in Allpahuayo National Reserve and Jenaro Herrera community (Loreto – Peru). Based on traditional use, ethanolic extracts were tested in vitro on different parasites strain (Plasmodium falciparum and Leishmania donovani) [2,3]. Piper amazonicum (Miq.) C. DC. (Piperaceae) and Grias neuberthii J.F.Macbr. (Lecythidaceae) showed strong activity (< 1 µg/mL) against Plasmodium falciparum 3D7 chloroquine-sensible strain and < 10 µg/mL against Leishmania donovani (intramacrophagic stage in LV 9 strain), along with low cytotoxicity on macrophages (> 25 µg/mL). In order to determine the anti-malaria mechanism of action, heme binding method by mass spectroscopy (MS) associated to molecular networking interpretation were investigated. Chemical profiling by LC-SPE-NMR of these extracts were performed which allowed the rapid identification of the main active compounds and major known metabolites while avoiding tedious purification procedures [4]. 2 new compounds from P. amazonicum (C₁₃H₁₈N₂O₆ and C₂₆H₁₆N₂O₁₁) and 2 new compounds from G. neuberthii (C₃₇H₆₈N₅O₆ and C₄₀H₇₉N₄O₁₁) are being isolated as the antiplasmodial compounds. The study shows an example of fast targeting of active antiparasitic compounds from small amount of biological material (5 g of plant powder), allowing pharmacological exploration without threatening biodiversity. Acknowledgements: Consejo Nacional de Ciencia y Tecnología (CONCYTEC) is acknowledged for PhD funding of Pedro Vásquez-Ocmín. Keywords: Traditional medicine, chemical profiling, malaria, leishmania. References: [1] Zofou D, Nyasa RB, Nsagha DS, Ntie-Kang F, Meriki HD, Assob JCN, Kuete V. Control of malaria and other vector-borne protozoan diseases in the tropics: enduring challenges despite considerable progress and achievements. Infect Dis Poverty 2014; 3: 1 – 14 [2] Desjardins RE, Canfield CJ, Haynes JD, Chulay JD. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 1979; 16: 710 – 718 [3] Balaraman K, Campos VN, Moussa F, Vacus J, Cojean S, Pomel S, Bories C, Figadère B, Kesavan V, Loiseau P. In vitro and in vivo antileishmanial properties of a 2-n-propylquinoline hydroxypropyl b-cyclodextrin formulation and pharmacokinetics via intravenous route. Biomed Pharmacother 2015; 76: 127 – 133 [4] Hubert J, Nuzillard JM, Renault JH. Dereplication strategies in natural product research: How many tools and methodologies behind the same concept? Phytochem Rev 2015; 1 – 41

Eleven compounds were isolated from Poraqueiba sericea stems and identified as niga-ichigoside-F1 (1), trachelosperoside B1 (2), 4-epi-niga-ichigoside (7), 19α-hydroxyasiatic acid (3), myrianthic acid (4), hyptatic acid (5), trachelosperogenin B (6), arjunolic acid (8), and trachelosperogenin E (9), secologanoside (10) and secoxyloganin (11). Compounds 1–11 were tested for their antileishmanial activities against Leishmania infantum promastigotes, 1–6 and 8–11 were tested for their cytotoxic activities on fibroblasts, 1–3, 5–6, 8–11 were evaluated for their anti-elastase and anti-acetylcholinesterase assays activities by a spectrophotometric method and 1–2, 5 and 7–10 were tested using bioautography for their β-glucosidase. No antileishmanial activity was detected; compounds 1, 2 and 11 showed a moderate cytotoxic activity with IC₅₀ 17.7, 20.5 and 10.9 μg/mL, respectively; compounds 2, 8, 9 and 10 gave a percentage of inhibition ranging from 13 to 16% (at 50 μg/mL) and compounds 1 and 2 showed an inhibition zone on β-glucosidase and anti-acetylcholinesterase assays.