Búsqueda de compuestos con posible actividad insecticida a partir de cianobacterias del Caribe colombiano

Abstract

Se estudiaron tres mats de cianobacterias usando fraccionamiento bioguiado con el ensayo larvicida contra Aedes aegypti. Como compuestos activos se aislaron ácidos grasos insaturados, dos nuevos análogos de dolastatina D y el ácido seco malyngólido (nuevo), también la neodysidenina que afectó el desarrollo larvario y un heptapéptido cíclico nuevo mostró actividad promisoria.

Abstract: Aedes aegypti L. [Diptera: Culicidae] is the disease vector mosquitoe for dengue, chikungunya, zika and yellow fever. Recently, several epidemic outbreaks occurred in hydroxyisovaleric acid)-(2-methyl-3-aminobutanoic acid)-(Ileu)-(2-hydroxy-3- phenilpropanoic acid)) CB1 and cyclo(NMe-(Ala)-(2-hydroxyisovaleric acid)-(2- methyl-3-aminobutanoic acid)-(Leu)-(2-hydroxy-3-phenilpropanoic acid)) CB2. These peptides are new dolastatin D analogs. CB1 and CB2 showed a moderated larvicide bioactivity (58% mortality, 0.09 mM), low ecotoxicity (low A. saline mortality) and were not AChE inhibitors. In addition, miristic acid (1), palmitic acid (2) and methyl palmitate (3) were identified by GC-MS from FI and FII fractions. Only compound 1 exhibited a moderated larvicide activity, but resulted to be toxic for A. saline. A second cyanobacteria mat identifyed as Lyngbya sp. (IBUN-02213) was also studied. From the active BuOH fraction (FB) a single compound was identified as (2R,5S)- malyngolide seco acid (CB3) by NMR and LC-(+)-HRESIMS data. This compound was previously obtained as a product of malyngolide basic hydrolysis, but was never reported as a natural product before. Then, this is the first time malyngolide seco acid is isolated as a secondary metabolite. In addition, malyngolide (CB4) was also isolated from this mat FD fraction. Malyngolide mat producer could be a new cyanobacterial gender among Lyngbya gender taxonomical reclassification. Compounds 1-3, methyl miristate (4), heptadecene (5) and phytone (6) were identified by GC-MS from FD. Compound 7 1-acetoxy-3-acetoxymethyl-7,11,15-trimethylhexadec-2,6,10,14- tetraene was detected by LC-HRESIMS of the same mat. The bioactivity assays for these compounds showed a moderated larvicide activity of CB3 along with a low toxicity against A. salina and probably AChE inhibition is its action mode. CB4 was not larvicide but showed a high A. saline lethality. This suggest that CB3 could be a good larvicida with no ecotoxic effects and it is obtained in high yields. Finally, the FD fraction of Moorea producens mat (IBUN-03496) exhibited a high larvae mortality (100% at 50 μg/well and 30% at 10 μg/well). Chemical analysis by GC-MS allowed identifing compounds 1-4 (also isolated from IBUN-02213), methyl stearate (8), methyl palmitoleate (9), mehtyl oleate (10) and crinasterol (12). Cholesterol (11), clionasterol (13) and campesterol (14) were isolated and elucidated by NMR and GCMS analysis. Additionally, a cis monounsaturated fatty acid showed a potent larvicide Colombia and worldwide. Control strategies focused on vector larvae control and are a global concern for these diseases prevention. Therefore, in this study, our aim was to isolate larvicide compounds against Ae. aegypti from Colombian Caribbean cyanobacterial mats. As a first step, 16 cyanobacterial mats were extracted with DCM/MeOH (1:1). Then, crude extract was fractionated by L-L partition in order to obtain a dichlorometane (FD) and buthanol (FB) fractions. All FD and FB fractions were evaluated in 3 different bioassays: larvicide assay against Ae. aegypti; ecotoxicity assay against Artemia salina and acetylcholinesterase inhibition assay as an approach to describe the possible action mode. In general terms, FD fractions showed to be more active than FB fractions. In addition, mats from the same species collected in different years exhibited different larvicide bioactivity (i.e. Phormidium submembranaceum /Symploca hynoides mats), as well as the ones that were collected at the same time in different locations (i.e. Moorea producens mats). As a result, three mats were selected for chemical studying according to bioassays results. A cyanobacterial mat identifyed as Phormidium sp. (IBUN-02224) was fractionated using a DIOL cartridge (mixtures hexane/ethyl acetate/MeOH, FI-FV), and FIII fraction showed larvicide activity along with a low letalithy over A. salina nauplii. This suggest that this fraction could control larvae with no ecotoxical effects. On the other hand, FIII did not show acetylcholinesterase inhibition, which is an advantage because insects have already developed insecticide resistance by AChE modification. FIII fraction purification yielded two new cyclic depsypeptides identifyed as cyclo(NMe-(Ala)-(2-hydroxyisovaleric acid)-(2-methyl-3-aminobutanoic acid)-(Ileu)-(2-hydroxy-3- phenilpropanoic acid)) CB1 and cyclo(NMe-(Ala)-(2-hydroxyisovaleric acid)-(2- methyl-3-aminobutanoic acid)-(Leu)-(2-hydroxy-3-phenilpropanoic acid)) CB2. These peptides are new dolastatin D analogs. CB1 and CB2 showed a moderated larvicide bioactivity (58% mortality, 0.09 mM), low ecotoxicity (low A. saline mortality) and were not AChE inhibitors. In addition, miristic acid (1), palmitic acid (2) and methyl palmitate (3) were identified by GC-MS from FI and FII fractions. Only compound 1 exhibited a moderated larvicide activity, but resulted to be toxic for A. saline. A second cyanobacteria mat identifyed as Lyngbya sp. (IBUN-02213) was also studied. From the active BuOH fraction (FB) a single compound was identified as (2R,5S)- malyngolide seco acid (CB3) by NMR and LC-(+)-HRESIMS data. This compound was previously obtained as a product of malyngolide basic hydrolysis, but was never reported as a natural product before. Then, this is the first time malyngolide seco acid is isolated as a secondary metabolite. In addition, malyngolide (CB4) was also isolated from this mat FD fraction. Malyngolide mat producer could be a new cyanobacterial gender among Lyngbya gender taxonomical reclassification. Compounds 1-3, methyl miristate (4), heptadecene (5) and phytone (6) were identified by GC-MS from FD. Compound 7 1-acetoxy-3-acetoxymethyl-7,11,15-trimethylhexadec-2,6,10,14- tetraene was detected by LC-HRESIMS of the same mat. The bioactivity assays for these compounds showed a moderated larvicide activity of CB3 along with a low toxicity against A. salina and probably AChE inhibition is its action mode. CB4 was not larvicide but showed a high A. saline lethality. This suggest that CB3 could be a good larvicida with no ecotoxic effects and it is obtained in high yields. Finally, the FD fraction of Moorea producens mat (IBUN-03496) exhibited a high larvae mortality (100% at 50 μg/well and 30% at 10 μg/well). Chemical analysis by GC-MS allowed identifing compounds 1-4 (also isolated from IBUN-02213), methyl stearate (8), methyl palmitoleate (9), mehtyl oleate (10) and crinasterol (12). Cholesterol (11), clionasterol (13) and campesterol (14) were isolated and elucidated by NMR and GCMS analysis. Additionally, a cis monounsaturated fatty acid showed a potent larvicide activity. Two peptides named neodysidenin (CB5) and the novel 10- dechloroneodysidenine (CB6) were isolated and their structures were proposed by NMR and LC-(+)-HRESIMS. From the most active fraction, a new compound CB8 (1mg) was isolated. It was identified as a cyclic depsipeptide cyclo((Dhoya)-(Thr)-NMe- (OMe-Tyr)-(Val)-(3,5-diaminopentanoic acid)-NMe-(Ileu)-(Leucic acid)). Dhoya is a very uncommon hydroxiacid that has been previously reported in cianobacterial compounds, and β-aminoacid 3,5-diaminopentanoic acid is found in a marine natural product for the first time. Regarding the results in the bioactivity tests monounsaturated fatty acids, as oleic acid, exhibited potent larvicide activity with low A. saline toxicity and no activity in AChE inhibition test. Neodysidenina showed a low larvicide activity but a larvae development disruption was observed. This research work shows the high biodiversity of cyanobacterial compounds with potencial use as larvicides controllers. Additonally, this thesis results contribute to chemiotaxonomy of Caribbean cyanobacterial mats, showing the importance of this taxonomical approach in biorpospection studies.