In vitro ovicidal and larvicidal activities of some saponins and flavonoids against parasitic nematodes of goats.

This study assessed the anthelmintic activity of plant-derived compounds against gastrointestinal nematodes of goats using the egg hatch and larval motility assays. The compounds tested were saponins (digitonin and aescin) and their respective sapogenins (aglycones), hecogenin acetate and flavonoids (catechin, hesperidin, isocordoin and a mixture of isocordoin and cordoin). Additionally, cytotoxicity of active substances was analysed on Vero cell through 3-4,5-dimethylthiazol-2-yl,2,5diphenyltetrazolium bromide (MTT) and propidium iodide (PI) tests. Significant reduction on the egg hatching (P 90%). Nevertheless, higher cytotoxicity was observed in the MTT assay, with IC50 of 0.20 mg mL-1 (aescin) and 0.0074 mg mL-1 (digitonin). Aescin and digitonin have a pronounced in vitro anthelmintic effect and the glycone portion of these saponins plays an important role in this activity.

Homology modeling, docking, molecular dynamics and in vitro studies to identify Rhipicephalus microplus acetylcholinesterase inhibitors.

Rhipicephalus microplus is an important ectoparasite of cattle, causing considerable economical losses. Resistance to chemical acaricides has stimulated the search for new antiparasitic drugs, including natural products as an eco-friendly alternative of control. Flavonoids represent a class of natural compounds with many biological activities, such as enzyme inhibitors. Acetylcholinesterase is an essential enzyme for tick survival that stands out as an important target for the development of acaricides. This work aimed to predict this 3D structure by homology modeling and use the model to identify compound with inhibitory activity. The model of R. microplus AChE1 (RmAChE1) was constructed using MODELLER program. The optimization and molecular dynamic investigation were performed in GROMACS program. The model developed was used, by molecular docking, to evaluate the anticholinesterase activity of flavonoids (quercetin, rutin, diosmin, naringin and hesperidin) and an acaricide synthetic (eserine). Additionally, in vitro inhibition of AChE and larval immersion tests were performed. The model of RmAChE1 showed to be sterically and energetically acceptable. In molecular dynamics simulations, the 3D structure remains stable with Root Mean Square Deviation = 3.58 Å and Root Mean Square Fluctuation = 1.43 Å. In molecular docking analyses, only eserine and quercetin show affinity energy to the RmAChE (Gridscore: -52.17 and -39.44 kcal/mol, respectively). Among the flavonoids, quercetin exhibited the best in vitro inhibition of AChE activity (15.8%) and mortality of larvae tick (30.2%). The use of in silico and in vitro techniques has shown that quercetin showed promising anti-tick activity and structural requirements to interact with RmAChE1. Communicated by Ramaswamy H. Sarma.

In vitro anthelmintic evaluation of three alkaloids against gastrointestinal nematodes of goats.

This study assessed the in vitro anthelmintic activity of the alkaloids berberine, harmaline and piperine on gastrointestinal nematodes (GIN) of goat and their possible cytotoxic effects in Vero cells. The anthelmintic evaluation was performed using the egg hatch (EHA) and larval motility (LMA) assays. Cytotoxicity was determined using the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) assay. The alkaloids berberine and piperine inhibited the hatching of GIN eggs in more than 90 %. Piperine was the most active compound against goat GIN eggs with an EC50 (effective concentration 50 %) of 0.0074 mM (0.0021 mg/mL), while the EC50 of berberine was 1.32 mM (0.49 mg/mL). Harmaline (EC50 = 1.6 mM - 0.34 mg/mL) showed moderate ovicidal action (80.30 %). In LMA, piperine and harmaline reduced larval motility in 2.75 and 25.29 %, respectively. Larvicidal efficacy was evidenced only with the alkaloid berberine, which showed a percentage of inhibition of larval motility of 98.17 % (2.69 mM =1.0 mg/mL). In the MTT assay, all alkaloids showed low toxicity to Vero cells, with a percentage of cell viability greater than 50 % in all concentrations tested. These results suggest that berberine and piperine have anthelmintic potential on goat gastrointestinal nematodes with low toxicity to mammalian cells.

In vitro and in silico studies of the larvicidal and anticholinesterase activities of berberine and piperine alkaloids on Rhipicephalus microplus.

Rhipicephalus microplus is responsible for high economic losses in livestock and its control has become difficult due to the establishment of tick populations resistant to commercial acaricides. This study aimed to evaluate the in vitro larvicidal effect of the alkaloids berberine and piperine, and also to investigate their inhibitory mechanisms against the acetylcholinesterase enzyme. The effects of the alkaloids on larvae were observed through the immersion test at the following concentrations: 1.5; 3; 6; 12; 16 and 24 mM. Berberine and piperine presented larvicidal activity greater than 95 %, not differing from 100 % for the positive fipronil control (p > 0.05). Of the two alkaloids, piperine had a lower effective concentration (EC), with an EC50 of 6.04 mM. The acetylcholinesterase enzyme used in the study was obtained from R. microplus larvae (RmAChE) and the anticholinesterase activity was determined spectrophotometrically. The highest anticholinesterase activity, measured as inhibition concentration (IC), was observed for berberine (IC50 = 88.13 µM), while piperine showed lower activity (IC50 > 200 µM). Docking studies in RmAChE, followed by 10 ns molecular dynamics simulation, suggest that berberine stabilizes the RmAChE at lower Root-Mean-Square Deviation (RMSD) than Apo protein. Few hydrogen-bond interactions between berberine and RmAChE residues were balanced by hydrophobic and π-type interactions. Berberine fills preferentially the peripheral anionic site (PAS), which correlates with its non-competitive mechanism. These results suggest that berberine and piperine alkaloids have an in vitro acaricidal action on R. microplus larvae, and the likely mechanism of action of berberine is related to RmAChE inhibition when accessing the PAS residues. These findings could help the study of new natural products that could inhibit RmAChE and aid in the development of new acaricides.

Assessment of in vitro anthelmintic activity and bio-guided chemical analysis of BRS Boyrá pineapple leaf extracts.

Species of the Bromeliaceae are known for their pharmacological actions, including anthelmintic effects. The aim of this study was to investigate the in vitro anthelmintic activity of extracts and fractions of BRS Boyrá pineapple leaf against the eggs and infective larvae of gastrointestinal nematodes (Trichostrongylidae) of goats and to identify the compounds involved in this activity. Crude methanol, hexane, dichloromethane, ethyl acetate and residual hydromethanol extracts were investigated by quantitative analysis of phenolic and flavonoid contents, antioxidant activity, anthelmintic activity against gastrointestinal nematodes of goats. The extracts were submitted to chromatographic methods for substance isolation and spectrometric techniques to identify their structures. The anthelmintic activity was performed by in vitro assays with eggs and larvae of nematodes obtained from naturally infected donor goats. All extracts contained phenolic (2.22-14.12 g of gallic acid equivalent per 100 g of dry extract) and flavonoid compounds (0.13-1.45 g of quercetin equivalent per 100 g of dry extract). Bio-guided fractionation of the BRS Boyrá pineapple leaves showed high antioxidant activity (EC50 for DPPH of 2.16-21.38 mg mL-1 and inhibition of co-oxidation of ß-carotene of 36.40-74.86%) and anthelmintic activity (15.69-100% inhibition of egg hatching). The ethyl acetate extract exhibited greatest activity in all assays. Through chromatographic column analysis it was possible to isolate three substances: ß-sitosterol and stigmasterol mixture in dichloromethane and hexane extracts, identified by NMR and p-coumaric acid in the ethyl acetate extract, identified by HPLC-DAD. The isolated p-coumaric acid exhibited high ovicidal effect against goat gastrointestinal nematodes (IC50: 0.12 mg mL-1) and can be considered the active substance of the ethyl acetate extract. This study revealed for the first time that the pineapple BRS Boyrá possesses inhibitory activity against gastrointestinal nematodes (Haemonchus spp., Oesophagostomum spp. and Trichostrongylus spp.), and that p-coumaric acid is an important bioactive.

Anti-tick effect and cholinesterase inhibition caused by Prosopis juliflora alkaloids: in vitro and in silico studies.

We investigated the in vitro acaricide activity of the methanolic extract (ME) and alkaloid-rich fraction (AF) of Prosopis juliflora on Rhipicephalus microplus and correlated this effect with acetylcholinesterase (AChE) inhibition. The acaricide activity was evaluated using adult and larval immersion tests. Also, we studied the possible interaction mechanism of the major alkaloids present in this fraction via molecular docking at the active site of R. microplus AChE1 (RmAChE1). Higher reproductive inhibitory activity of the AF was recorded, with effective concentration (EC50) four times lower than that of the ME (31.6 versus 121 mg/mL). The AF caused mortality of tick larvae, with lethal concentration 50% (LC50) of 13.8 mg/mL. Both ME and AF were seen to have anticholinesterase activity on AChE of R. microplus larvae, while AF was more active with half-maximal inhibitory concentration (IC50) of 0.041 mg/mL. The LC-MS/MS analyses on the AF led to identification of three alkaloids: prosopine (1), juliprosinine (2) and juliprosopine (3). The molecular docking studies revealed that these alkaloids had interactions at the active site of the RmAChE1, mainly relating to hydrogen bonds and cation-pi interactions. We concluded that the alkaloids of P. juliflora showed acaricide activity on R. microplus and acted through an anticholinesterase mechanism.

In vitro acaricide and anticholinesterase activities of digitaria insularis (Poaceae) against Rhipicephalus (Boophilus) microplus.

Medicinal plants have been proposed as an alternative for acaricide control, aiming to develop lower-cost and eco-friendly ectoparasiticide products. The aim of this study was to evaluate the in vitro activity of the extracts and fractions obtained from the leaves of Digitaria insularis on the reproductive efficacy of the bovine tick Rhipicephalus (Boophilus) microplus. Also, we investigated the possible relation with the anticholinesterase mechanism. The effect of the crude hydroethanolic (CH), hexanic (HE), ethyl acetate (EA), butanolic (BT) and residual hydroethanolic (RH) extracts, as well as four fractions of HE, were evaluated using adult immersion test. Only the HE and EA extracts (50 mg/mL) and fraction 2 (Fr2) (12.5 mg/mL) promoted reduction of the reproductive parameters (oviposition and hatching rate) greater than 90% and were not statistically different from the positive control. Higher reproductive activity was recorded in Fr2 with a lower effective concentration (EC50) value (6.65 mg/mL) than in HE (17.8 mg/mL) and EA (23.97 mg/mL). The anticholinesterase activity was assessed through spectrophotometry in microtiter assays, with enzymatic inhibition of 34.8, 43.2 and 57.9% of the HE, AE and Fr2, respectively. The chemical evaluation of the Fr2 was carried through Gas Chromatography coupled to Mass Spectrometry (GC-MS) and led to the characterization of nine compounds classified as fatty acids (3), esterified fatty acids with long-chain alcohol (4) and terpene (1). The effect of D. insularis extracts and fractions was focused on female reproductive parameters such as oviposition and hatching rates. The results obtained in this study suggest that D. insularis shows an in vitro acaricidal activity against R. (B.) microplus. Such action might be associated with the presence of secondary metabolites identified in the Fr2.

Prosopis juliflora Pods Alkaloid-rich Fraction: In vitro Anthelmintic Activity on Goat Gastrointestinal Parasites and Its Cytotoxicity on Vero Cells.

BACKGROUND: This study was designed to assess the in vitro anthelmintic activity of the fraction containing alkaloid from Prosopis juliflora pods on goat gastrointestinal nematodes using the egg hatch assay (EHA), larval migration inhibition assay (LMIA), and larval motility assay (LMA). MATERIALS AND METHODS: The alkaloid-rich fraction (AF) - content juliprosopine as major alkaloid - was obtained from ethyl acetate extract after fractionation in Sephadex LH-20 chromatography column and its characterization were made by nuclear magnetic resonance analysis together with literature data comparison. The concentrations tested were 4.0, 2.67, 1.78, 1.19, and 0.79 mg/mL (EHA) and 4 mg/mL (LMIA and LMA). The in vitro cytotoxicity on Vero cell cultures was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue tests. RESULTS: High ovicidal activity was observed with IC50 and IC90 values at 1.1 and 1.43 mg/mL for AF. On the other hand, this fraction showed low larvicidal activity and high toxic effect. CONCLUSION: Thus, P. juliflora pod alkaloid rich-fraction has ovicidal activity in vitro against goat gastrointestinal nematodes and cytotoxic in Vero cell cultures. SUMMARY: Prosopis juliflora alkaloid-rich fraction (AF) showed in vitro anthelmintic effect against gastrointestinal nematodes of goatsThe AF was more effective against eggs than third larval stage (L3) of gastrointestinal nematodesThe AF showed cytotoxicity activity on Vero cell lineThe juliprosopine was the main alkaloid found in the AF from P. juliflora pods. Abbreviations used: AF: Alkaloid-rich fraction; DMSO: Dimethyl sulfoxide; EE: Ethyl acetate extract; EHA: Egg hatch assay; IC50: Inhibitory concentration 50%; IC90: Inhibitory concentration 90%; L3: Infective larvae; LMA: Larval motility assay; LMIA: Larval migration inhibition assay; MTT: Bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMR: Nuclear magnetic resonance; PBS: Phosphate buffered saline; RPMI: Roswell Park Memorial Institute médium; TLC: Thin Layer Chromatography.

In vitro anthelmintic and cytotoxicity activities the Digitaria insularis (Poaceae).

This study aimed to evaluate the in vitro activity of D. insularis extracts and fractions against gastrointestinal nematodes of goats and its cytotoxicity on Vero cells. The egg hatch (EHT) and larval motility (LMT) tests were conducted to investigate the anthelmintic effects of the crude hydroethanolic (CH), ethyl acetate (EA), butanolic (BT) and residual hydroethanolic (RH) extracts. The elution of the active extract (EA) on column chromatography (SiO2) using organic solvents furnished six fractions (FR1 to FR6), which were also tested. Cytotoxicity was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan Blue exclusion assays. All extracts, FR2 and FR3, inhibited egg hatching in a concentration-dependent manner. The EHT led to EC50 values (effective concentration 50%) of 0.64; 0.69; 0.77; 0.96; 0.27 and 0.65mg/mL for CH, EA, BT, RH, FR2 and FR3, respectively. However, the extracts exhibited low effect on the motility of L3. In the cytotoxicity evaluation (MTT assay), the IC50 (inhibitory concentration 50%) was 1.18 (EA), 1.65 (FR2) and 1.59mg/mL (FR3), which was relatively high (low toxicity) in comparison to the EC50 values in EHT, mainly for FR2. The chemical analyses of most active fractions (FR2) by Liquid Chromatography coupled to Mass Spectrometry (LC-MS) led the characterization of the flavones tricin and diosmetin. These results showed the high anthelmintic effect and low cytotoxicity of D. insularis and also that the flavones can be probably responsible for the nematocidal activity of this plant.

In vitro anthelmintic activity of the Zizyphus joazeiro bark against gastrointestinal nematodes of goats and its cytotoxicity on Vero cells.

This study examined the in vitro effect of the Zizyphus joazeiro bark against gastrointestinal nematodes of goats and its cytotoxicity on Vero cells. The ovicidal activity of the crude hydroethanolic extract (CE), its partitioned hexane (HE) and aqueous extract (AE) and saponins fraction (SF), including betulinic acid (BA), a biogenetic compound from this plant found in HE, were investigated using the inhibition of egg hatch assay (EHA). Thereafter, the extracts and the SF were evaluated through the larval motility assay (LMA) and larval migration inhibition assay (LMIA). The AE and SF promoted a complete inhibition of the egg hatch, and the effective concentration to inhibit 50% (EC50) values was 1.9 and 1.3mg/mL, respectively. The highest percentages of inhibition in EHA observed after treatments with CE, HE and BA corresponded to 79, 48 and 17%, respectively. The extracts and SF did not show larvicidal activity in LMA and LMIA. The AE and SF demonstrated cytotoxic effects in 3-4,5-dimethylthiazol-2-yl, 2,5diphenyltetrazolium bromide (MTT) and trypan blue tests; however, SF was more toxic (50% inhibitory concentration, IC50=0.20mg/mL). The chemical characterization of the SF was made through Proton Nuclear Magnetic Resonance ((1)H NMR) and Electrospray Ionization Mass Spectrometry (ESI-MS) analyses, which led to the identification of two saponins known as Joazeiroside B and Lotoside A. The results obtained from the research of this saponin content provide important information about the biological activity, especially the anthelmintic effect present in the plant investigated. That also suggests the types of bioactive compounds that may be responsible for this antiparasitic activity exhibited by the plant extracts.

Evaluation of anthelmintic activity of liquid waste of Agave sisalana (sisal) in goats.

It was evaluated the anthelmintic activity of Agave sisalana (sisal) juice against gastrointestinal nematodes and its potential toxic effects in goats. In vitro tests showed more than 95% reduction in larval counts of the genus Haemonchus spp. at concentrations between 86.5 and 146.3 mg.mL(-1). In vivo the percent reduction of larvae of the fourth (L4) and fifth (L5) stages of Haemonchus, Oesophagostomum and Trichostrongylus was less than 95% in groups GI and GII, and between 80 and 90% in group GIII. A. sisalana juice at the concentrations tested in vitro was effective against gastrointestinal nematodes in goats; however, its anthelmintic efficacy was reduced when administered to animals.

[Anthelmintic activity of aqueous extract of Zanthoxylum rhoifolium Lam. leaves (Rutaceae)]. / Atividade anti-helmíntica do extrato aquoso das folhas de Zanthoxylum rhoifolium Lam. (Rutaceae).

The study evaluated the anthelmintic activity of aqueous extract of Zanthoxylum rhoifolium leaves in two experiments. In vitro test, cultures of goat fecal samples were treated with different concentrations of extract (134.5 to 335.0 mg.mL(-1)). In vivo test was composed of 20 sheep: G1: treated with 0.63 g.kg(-1), during four days; G2: same dose, for eight days; G3: ivermectin (200 microg.kg(-1)) and G4 untreated group. In vitro results showed a reduction of Haemonchus spp, Trichostrongylus spp. and Oesophagostomum spp. larvae greater than 95% in the concentrations between 335.0 and 193.7 mg.mL(-1). Faecal egg counting reduction was 51, 56 and 90% in G1, G2 and G3, respectively, while immature stages and adults ranged from 0 to 91% in G1 and from 26 to 94% in G2. Ivermectin effectiveness was 99% for L4 and L5 of H. contortus and 100% for other nematodes species. Clinical and biochemical parameters have remained in the normality and histophatologic analyses did not show alteration suggesting absence of toxicity. Although the great effectiveness of Z. rhoifolium leaves extract in vitro test, it displayed poor efficiency in vivo regarding gastrointestinal nematodes reduction.

Evaluation of anthelmintic activity of liquid waste of Agave sisalana (sisal) in goats / Avaliação da atividade anti-helmíntica do resíduo líquido de Agave sisalana (sisal) em caprinos

It was evaluated the anthelmintic activity of Agave sisalana (sisal) juice against gastrointestinal nematodes and its potential toxic effects in goats. In vitro tests showed more than 95 percent reduction in larval counts of the genus Haemonchus spp. at concentrations between 86.5 and 146.3 mg.mL-1. In vivo the percent reduction of larvae of the fourth (L4) and fifth (L5) stages of Haemonchus, Oesophagostomum and Trichostrongylus was less than 95 percent in groups GI and GII, and between 80 and 90 percent in group GIII. A. sisalana juice at the concentrations tested in vitro was effective against gastrointestinal nematodes in goats; however, its anthelmintic efficacy was reduced when administered to animals.

Foi avaliada a atividade anti-helmíntica do suco de Agave sisalana (sisal) contra nematódeos gastrintestinais e possíveis efeitos tóxicos em caprinos. Nos testes in vitro, encontrou-se redução superior a 95 por cento na contagem de larvas do gênero Haemonchus spp. nas concentrações entre 86,5 e 146,3 mg.mL-1. In vivo, o percentual de redução de larvas de quarto (L4) e quinto (L5) estágios de Haemonchus, Oesophagostomum e Trichostrongylus foi inferior a 95 por cento para o GI e GII, e entre 80 e 90 por cento para o GIII. O suco de A. sisalana nas concentrações testadas in vitro foi efetivo contra nematódeos gastrintestinais de caprinos, apresentando, no entanto, reduzida eficácia anti-helmíntica quando administrado nos animais.

Atividade anti-helmíntica do extrato aquoso das folhas de Zanthoxylum rhoifolium Lam. (Rutaceae) / Anthelmintic activity of aqueous extract of Zanthoxylum rhoifolium Lam. leaves (Rutaceae)

O estudo avaliou a atividade anti-helmíntica do extrato aquoso das folhas de Zanthoxylum rhoifolium em dois experimentos. O teste in vitro utilizou cultivos de fezes de caprinos tratados com diferentes concentrações do extrato: (134,5 a 335,0 mg.mL-1). No teste in vivo, utilizaram-se 20 ovelhas: G1 tratado durante quatro dias (0,63 g.kg-1 PV); G2, mesma dose, por oito dias; G3, ivermectina (200 μg.kg-1); e G4, sem tratamento. In vitro, observou-se redução de larvas de Haemonchus, Trichostrongylus e Oesophagostomum superior a 95% nas concentrações de 193,7 a 335,0 mg.mL-1. In vivo, a redução de ovos por grama de fezes foi de 51, 56 e 90%, respectivamente, para G1, G2 e G3, no oitavo dia de tratamento, enquanto para os estágios imaturos e adultos variou de 0 a 91% no G1 e 26 a 94% no G2. A eficácia da ivermectina alcançou 99% para L4 e L5 de H. contortus e 100% para as demais espécies de nematoides. Parâmetros clínicos e bioquímicos permaneceram na faixa de normalidade, e as análises histopatológicas não revelaram alterações sugestivas de toxicidade. Embora altamente efetivo in vitro, o extrato aquoso de folhas de Z. rhoifolium foi pouco eficaz in vivo na redução de nematoides gastrintestinais.

The study evaluated the anthelmintic activity of aqueous extract of Zanthoxylum rhoifolium leaves in two experiments. In vitro test, cultures of goat fecal samples were treated with different concentrations of extract (134.5 to 335.0 mg.mL-1). In vivo test was composed of 20 sheep: G1: treated with 0.63 g.kg-1, during four days; G2: same dose, for eight days; G3: ivermectin (200 μg.kg-1) and G4 untreated group. In vitro results showed a reduction of Haemonchus spp, Trichostrongylus spp. and Oesophagostomum spp. larvae greater than 95% in the concentrations between 335.0 and 193.7 mg.mL-1. Faecal egg counting reduction was 51, 56 and 90% in G1, G2 and G3, respectively, while immature stages and adults ranged from 0 to 91% in G1 and from 26 to 94% in G2. Ivermectin effectiveness was 99% for L4 and L5 of H. contortus and 100% for other nematodes species. Clinical and biochemical parameters have remained in the normality and histophatologic analyses did not show alteration suggesting absence of toxicity. Although the great effectiveness of Z. rhoifolium leaves extract in vitro test, it displayed poor efficiency in vivo regarding gastrointestinal nematodes reduction.

Efeitos do suco de alho (Allium sativum Linn.) sobre nematódeos gastrintestinais de caprinos / Effects of garlic juice (Allium sativum Linn.) on gastrintestinal nematodes of goats

O objetivo deste trabalho foi avaliar os efeitos do suco de alho sobre nematódeos gastrintestinais de caprinos. Foram utilizados 20 animais, distribuídos em dois grupos tratados com o suco de alho, um grupo tratado com ivermectina e um grupo controle sem tratamento. Os percentuais de redução do número de ovos e larvas de Strongyloidea foram inferiores a 95 por cento para todos os grupos. O tratamento de caprinos com o suco de alho não foi eficiente no controle de nematódeos gastrintestinais.

Cytotoxicity effect of alkaloidal extract from Prosopis juliflora Sw. D.C. (Algaroba) pods on glial cells

Prosopis juliflora is largely used for feeding cattle and humans. Neurological signals have been reported in cattle due to intoxication with this plant. In this study, an alkaloidal fraction (AF) obtained from P. juliflora pods was tested on astrocyte primary cultures. Astrocytes display physiological functions essential to development, homeostasis and detoxification in the central nervous system (CNS). These cells are known for their role on energetic support and immune response in the CNS. Concentrations between 0.03 to 30 µg/ml AF were assayed for 24 - 72 h. The mitochondrial activity, assayed by MTT test, showed cytotoxicity at 30 µg/ml AF after 24 h. At concentrations ranging between 0.3 - 3 µg/ ml, the AF induced an increase on mitochondrial activity, indicating cell reactivity. lmmunocytochemistry assay for GFAP cytoskeletal protein, revealed alterations on cell morphology after treatment with 0.3 - 3 µg/ ml AF for 72 h. This result corroborates with western blot analysis when ceUs treated with 0.3 - 3 I-µg/ml AF for 72 h showed GFAP upregulation. The vimentin expression was not significantly altered in all tested concentrations. These results suggest that alkaloids induce astrocyte reactivity and might be involved in the neurotoxic effects induced by P. juliflora consumption.

A Prosopis juliflora é amplamente utilizada na alimentação humana e de várias espécies animais, especialmente bovinos. Quadros de intoxicação por esta planta, nesta espécie, têm sido relatados, principalmente quando a mesma é oferecida como única fonte alimentar, desencadeando uma doença de sintomatologia nervosa. Neste estudo, objetivou-se avaliar os efeitos in vitro da fração de alcalóides totais (F A) extraída das vagens da Prosopis juliflora utilizando cultura primária de astrócitos obtidos do córtex cerebral de ratos como modelo de estudo. A avaliação da atividade mitocondrial pelo teste do MTT demonstrou a citotoxicidade em 30 µg/ ml da FA após 24 h. As concentrações de 0,3 e 3 µg/ ml da FA induziram um aumento da atividade mitocondrial, indicando reatividade celular. Testes imunocitoquimicos para a GFAP, principal proteína de citoesqueleto de astrócitos, revelaram alterações morfológicas nas células após tratamento com 0,3 e 3 µg/ml da FA por 72 h. Tais resultados são consoantes à análise desta proteína por westernblot, quando as culturas foram tratadas com 0,3 e 3 µg/ml da FA por 72 h, demonstrando interferências na regulação da expressão da GFAP. A expressão de vimentina não foi significativamente alterada em nenhuma das concentrações testadas. Estes resultados sugerem que os alcalóides da P.juliflora induzem a reatividade astrocitária, o que pode estar envolvido nos efeitos neurotóxicos providos pelo consumo desta planta.