Caracterização fitoquímica e neurofarmacológica de compostos inseticidas isolados de Prasiola crispa (Lightfoot) Kützing (1843)

Abstract

Organisms adapted to extreme environments such as Antarctica, tend to present a unique constitution in terms of secondary metabolites. Thus, studies aiming the elucidation of biological activity of vegetal organisms from these regions are relevant in terms of biotechnology. The aim of this work was to investigate the mechanism underlying the entomotoxic activity of Prasiola crispa seaweed from Antarctica mealting iced areas whole extract in neurophysiological models of Nauphoeta cinerea cockroaches. The mechanisms of the vegetal extract isolated bioactive compounds that was related to the entomotoxic activity were also studied. The seaweed was collected next to the Polish station Arctowski, at Almirantado bay. The toxic mechanism of the hexanic extract of Prasiola crispa, was initially demonstrated through toxicological assessments of lethality assays of LD50. Biochemical assays were also performed to determine the acetylcholinesterase (AChE) enzime activity on the cockroaches’ brain homogenate. It was also used in vivo and in situ biological models of cockroaches, in order to assess the extract’s activity on the different insect physiological systems, such as the cardiovascular system, the peripheral nervous system and the central nervous system. To accomplish this, we have used the cockroach in situ semi-isolated heart technique, the the coxal methatoraxic-abdutor nerve-muscle preparation in vivo and the measurement of grooming activity in vivo. The hexanic extract of Prasiola crispa (HPC) was prepared according to the conventional phytochemical technique. The phytochemical preliminar analysis was performed through thin layer chromatography (TLC). Thus, the assays to determine the minimum lethal dose (LD50) at 100, 200, 400 and 800 µg/g of animal weight, demonstrated that the dose of 400 µg/g induced 50% lethality, that was considered the LD50 for Nauphoeta cinerea, 24h after the adminstration of the compound (n= 10 in triplicate *p≤0,05). The assays for AChE activity showed that the HPC 100, 200, 400 and 800µg/g of animal weight, does not inhibit significantly this enzyme (p≥0.05, n=5 in triplicate). The biological activity of HPC 100, 200, 400 e 800 µg/g of animal weight on cockroach heart rate, showed a time-dependent cardiotoxic activity. When the HPC 100 µg/g was assayed, a nonsignificative positive chronotropic effect was observed. With the higher doses there were a progressive negative chronotropic effect that was maximum at 800 µg/g 35±3 beat./min; (n=6,*p≤0,05). The recordings of cockroach neuromuscular twich-tension with HPC 100, 200, 400 e 800 µg/g animal, showed a progressive dose and time-dependent neuromuscular blockade. Spontaneous twiches were detected 15-30min after HPC treatment in all experiments. In this assayes, HPC 800 µg/g induced the highest level of neuromuscular blockade 100% within 120min recordings (n=6, *p≤ 0,05). On these preparations the previous treatment with chloral hydrate 10µg/g animal, an N-methyl-d-aspartate blocker, increased the time for 50% neuromuscular blockade, and also inhibited the appearance of spontaneus twiches. The pretreatment with bicuculine 2.5 µg/g of animal weight a GABA receptor blocker, partially inhibited the neuromuscular blockade activity induced by HPC 800µg/g (n=6, p≤0,05). The animal grooming behavior analysis which is the leg and antennae cleaning behavior, also showed a time-dependent effect. In these assays the values for grooming in saline-treated animals was 153±8s/30min for the legs 75±5s/30min for the antennas, respectively. When DMSO 10% was assayed, no changes in the responses was observed in 10 the antenna grooming, and a slight but not significant decrease in the leg grooming was detected. HPC 100, 200, 400 and 800 µg/g of animal weight treatment induced a significant increase in the legs and antennas grooming, of 390 ±5s/30min for antenas and 395 ±5s/30min for legs, on the smallest tested dose (*p≤0,05). When higher HPC doses were assayed, there was a progressive decrease on grooming responses, that was maximum for antennas and legs and resulted in the extinction of grooming activity at 800μg/g dose (n=30, *p≤0.05). The determination of HPC chemical compounds by Nuclear Magnetic Rensonance (RMN), showed three crystals with high purity that were characterized as phytosterols: β-sitosterol (24α-etil-colest-5-enol) (m/z 414,71), campesterol (24α-metil-colest-5-enol) (m/z 400.68) e stigmasterol (24α-etil-colest- 5,22-dienol) (m/z 412,69). β-sitosterol was the most active compound in inducing neurotoxicity at the peripheral nervous system. Together, the results presented at this work demonstrated that Prasiola crispa hexanic extract induces entomotoxic activity at Nauphoeta cinerea model. This toxic effect would be prevalente on the peripheral nervous system. The changes induced on insect grooming activity demonstrate that the extract bioactive compounds are able to reach the insect central nervous system, inducing complex behavioral changes. The cardiotoxic activity was significant, and may contribute to the development of entomotoxic activity. The presence of phytosterols compounds in the extract composition may be related to the algae toxic activity. Future phytochemical assays for the determination of novel compounds in the extract may help in the elucidation of the extract entomotoxic activity.