Role of Synadenium grantii latex proteases in nematicidal activity on Meloidogyne incognita and Panagrellus redivivus / Papel das proteases do látex de Synadenium grantii na atividade nematicida sobre Meloidogyne incognita e Panagrellus redivivus

Abstract Synadenium grantii is a Euphorbiaceae plant commonly found in Brazil, known as Janaúba or Leitosinha, whose latex is traditionally used for several purposes. However, it is not known whether the nematicidal action of this plant latex occurs due to the action of proteases. The present work aims to evaluate the nematicidal activity of proteases from Synadenium grantii latex on Meloidogyne incognita and Panagrellus redivivus. S. grantii latex used in the present study was collected from specimens found in Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. The drained latex was collected in Eppendorf microtubes and immediately stored on ice at 4 °C. After this extraction, the latex was frozen (-20 °C) during 2 hours, thawed at room temperature (25 °C) and centrifuged at 10,000 g at 4 °C for 30 minutes to remove larger particles and concentrate the proteases. After the centrifugation, assays of enzymatic activity were performed in order to know in which of the phases the enzymes were found. S. grantii latex presented protease, but no chitinase activity. The results show that there was a significant difference (p <0.01) between the treated and control groups, with 100% mortality of Meloidogyne incognita and 72% average mortality of Panagrellus redivivus. In addition, it was demonstrated that the nematicidal action occurred due to the action of the proteases, since the control was only differentiated from the treatment by the presence of the enzymes with biological activity.

Resumo Synadenium grantii é uma planta Euphorbiaceae comumente encontrada no Brasil, conhecida como Janaúba ou Leitosinha, e tem seu látex usado tradicionalmente para diferentes propósitos. Entretanto, não se conhece se a atividade nematicida da planta ocorre devido à ação de proteases. O presente trabalho tem como objetivo avaliar a atividade nematicida das proteases do látex de Synadenium grantii sobre Meloidogyne incognita e Panagrellus redivivus. O látex de S. grantii utilizado no presente trabalho foi coletado a partir de espécimes encontradas na Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. O látex foi coletado em microtubos Eppendorf e imediatamente armazenado em gelo a 4 °C. Após esta extração, o látex foi congelado (-20 °C) durante 2 horas, descongelado à temperatura ambiente (25 °C) e centrifugado a 10000 g a 4 °C durante 30 minutos para a remoção de partículas e concentração das proteases. Após a centrifugação, foram realizados ensaios de atividade enzimática para saber em qual das fases as enzimas foram encontradas. O látex de S. grantii apresentou atividade de protease, mas nenhuma atividade de quitinase. Os resultados mostram que houve diferença significativa (p <0,01) entre os grupos tratados e controle, com 100% de mortalidade de Meloidogyne incognita e 72% de mortalidade média de Panagrellus redivivus. Além disso, foi demonstrado que a ação nematicida ocorreu devido à ação das proteases, uma vez que o grupo controle só foi diferenciado do tratamento pela presença das enzimas com atividade biológica.

Role of Synadenium grantii latex proteases in nematicidal activity on Meloidogyne incognita and Panagrellus redivivus

Abstract Synadenium grantii is a Euphorbiaceae plant commonly found in Brazil, known as Janaúba or Leitosinha, whose latex is traditionally used for several purposes. However, it is not known whether the nematicidal action of this plant latex occurs due to the action of proteases. The present work aims to evaluate the nematicidal activity of proteases from Synadenium grantii latex on Meloidogyne incognita and Panagrellus redivivus. S. grantii latex used in the present study was collected from specimens found in Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. The drained latex was collected in Eppendorf microtubes and immediately stored on ice at 4 °C. After this extraction, the latex was frozen (-20 °C) during 2 hours, thawed at room temperature (25 °C) and centrifuged at 10,000 g at 4 °C for 30 minutes to remove larger particles and concentrate the proteases. After the centrifugation, assays of enzymatic activity were performed in order to know in which of the phases the enzymes were found. S. grantii latex presented protease, but no chitinase activity. The results show that there was a significant difference (p 0.01) between the treated and control groups, with 100% mortality of Meloidogyne incognita and 72% average mortality of Panagrellus redivivus. In addition, it was demonstrated that the nematicidal action occurred due to the action of the proteases, since the control was only differentiated from the treatment by the presence of the enzymes with biological activity.

Resumo Synadenium grantii é uma planta Euphorbiaceae comumente encontrada no Brasil, conhecida como Janaúba ou Leitosinha, e tem seu látex usado tradicionalmente para diferentes propósitos. Entretanto, não se conhece se a atividade nematicida da planta ocorre devido à ação de proteases. O presente trabalho tem como objetivo avaliar a atividade nematicida das proteases do látex de Synadenium grantii sobre Meloidogyne incognita e Panagrellus redivivus. O látex de S. grantii utilizado no presente trabalho foi coletado a partir de espécimes encontradas na Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. O látex foi coletado em microtubos Eppendorf e imediatamente armazenado em gelo a 4 °C. Após esta extração, o látex foi congelado (-20 °C) durante 2 horas, descongelado à temperatura ambiente (25 °C) e centrifugado a 10000 g a 4 °C durante 30 minutos para a remoção de partículas e concentração das proteases. Após a centrifugação, foram realizados ensaios de atividade enzimática para saber em qual das fases as enzimas foram encontradas. O látex de S. grantii apresentou atividade de protease, mas nenhuma atividade de quitinase. Os resultados mostram que houve diferença significativa (p 0,01) entre os grupos tratados e controle, com 100% de mortalidade de Meloidogyne incognita e 72% de mortalidade média de Panagrellus redivivus. Além disso, foi demonstrado que a ação nematicida ocorreu devido à ação das proteases, uma vez que o grupo controle só foi diferenciado do tratamento pela presença das enzimas com atividade biológica.

Cytotoxicity of latex and pharmacobotanical study of leaves and stem of Euphorbia umbellata (Janaúba)

AbstractIn southern Brazil, the bottled latex of Synadenium grantii Hook f., Euphorbiaceae, is popularly used as a treatment of all types of cancer. Similarly, Synadenium umbellatum Pax. is used in the central western region of Brazil for the same purpose and in the same manner of use. Both plants are popularly known as janaúba or leitosinha. The objectives of this study were to use pharmacobotanical analysis to verify whether these two species, which are considered to be distinct, are actually the same to determine anatomical markers; to assist in the identification and differentiation of other Euphorbia; and to evaluate the cytotoxic activity of the latex in relation to HeLa and HRT-18 cells. Leaves and stems of the species were collected in Goiânia and Ponta Grossa and were investigated using scanning electron microscopy and optical microscopy techniques. The latex was also collected and analyzed in relation to its cytotoxic effect by employing MTT and NR techniques. The pharmacobotanical study of the specimens in both localities showed that they were the same species, namely Euphorbia umbellata (Pax) Bruyns, which is the scientific nomenclature accepted and confirmed by an expert taxonomist who specializes in Euphorbia. The pharmacobotanical characteristics highlighted in this study can assist in the identification of the taxon and contribute to the control of the quality of this plant drug. The evaluation of the latex in relation to HRT-18 cells demonstrated action after 48 h of experiment. In contrast, in relation to HeLa cells its induced cytotoxicity in all times and a dose-dependent manner. The IC50 values (72 h) observed were 252.58 ± 18.51 µg/ml and 263.42 ± 15.92 µg/ml to MTT experiment and 250.18 ± 19.48 µg/ml and 430.56 ± 19.71 µg/ml to NR experiment for the HeLa and HRT-18 cells, respectively.

Anti-ulcer activity of Synadenium grantii latex

Synadenium grantii Hook f., Euphorbiaceae, is popularly known as leitosinha or janaúba. The diluted latex (18 drops/L of water) is commonly used in the south of Brazil to treat gastric disturbances. This study evaluated phytochemical screening and toxicity using Artemia salina Leach of crude bark extract and also latex. The toxicity and the anti-ulcer activity of S. grantii latex were also tested in rats. Phytochemical results showed presence of tannins, terpenes, unsaponificable substances, coumarins and anthraquinones in the crude bark extract and terpenes in the latex. Gas chromatography-mass spectrometry (GC-MS) analysis demonstrated the presence of diterpene tigliane esters in the latex, identified as 12-deoxyphorbol-13-(2-metilpropionate) and phorbol 12,13,20-triacetate. The toxicity results using A. salina presented CL50 26.58μg/mL and CL50 778.66μg/mL, for the latex and the crude bark extract respectively. The toxicological hepatic parameters of the diluted latex were not different to the control group (p<0.05). The eosinophils cells showed an increase in both the diluted and pure latex groups. The pure latex showed gastric protection of 90% (p<0.05) and the diluted latex showed 6% compared to the negative control. Therefore, our data indicate that S. grantii latex, under research conditions presented gastric protection. Pure latex showed more toxicity than the diluted latex.

Eritrodermia secundaria a planta productora de látex (synadenium grantii) / Erythroderma secondary to latex-producing plants (synadenium grantii)

Las euforbiáceas son una familia de plantas que tienen en común la producción de látex. Existen más de 8000 especies.Tienen amplia distribución mundial, y adoptan la forma de árboles, arbustos o hierbas. Pertenecen a esta familia numerosas especies de importancia y, en nuestro país, se emplean con fines industriales y medicinales. También existen plantas ornamentales, cuya atractiva apariencia estimula la curiosidad de los niños, quienes pueden ingerirlas o manipularlas.En el Centro Nacional de Intoxicaciones, las consultas por plantas representan un escaso número (0,2 por ciento) dentro del total de accidentes en pediatría; sin embargo, pueden revestir gravedad.Presentamos el caso de una niña de 4 años, quien manipuló e ingirió parte de una planta de la familia Euforbiáceas(Synadenium grantii).

The Euphorbiaceae family are plants that have in common latex production. There are over 8000 species. They have worldwide distribution. We can find trees, shrubs or herbs. Many importantspecies belong to this family and they are used in our country for industrial and medical purposes. There are alsoornamental plants with attractive appearence that stimulate children curiosity, intake or manipulation.In the National Poison Center consultations for plants represent a small number (0.2%) of the total accidents in children, however they can be serious. We report the case of a 4 years old girl who manipulated andswallowed part of an Euphorbiaceae family plant (Synadenium grantii).

Purification and characterisation of a carboxylesterase from the latex of Synadenium grantii Hook,'f'.

The latex of Synadenium grantii was found to contain esterolytic activity. Polyacrylamide gel electrophoretic study coupled with substrate and inhibitor specificity studies revealed the presence of multiple forms of carboxylesterases and cholinesterases in the latex. One of the carboxylesterases of the latex was purified by acetone fractionation, carboxymethyl- Sephadex chromatography and Sepharose-6B gel filtration. The homogeneity of the enzyme was established by polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme consists of a single polypeptide chain with a molecular weight of 14,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to basic amino acid residues. The isoelectric pH of the enzyme was found to be 4·0. The enzyme was a glycoprotein as revealed by periodic acid Schiff-staining technique. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was sensitive to organophosphates. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear non-competitive inhibition with 1- naphthol.