Potencial anti-helmíntico de extratos proteicos de Leucaena leucocephala (Linn.) (Fabaceae) e Spigelia anthelmia (Linn.) (Loganiaceae) contra Haemonchus contortus (Rudolphi, 1803)

Abstract

Haemonchus contortus (RUDOLPHI, 1803) is a species of gastrointestinal nematode of great importance due to the damages caused in livestock. Natural products have been studied as an alternative to the use of commercial anthelmintics, responsible for the development of resistance in parasites. This study has as main objective to verify the anthelmintic activity of protein extracts of Leucaena leucocephala (Linn.) and Spigelia anthelmia (Linn.) against the nematode H. contortus. The present work was divided in two chapters. In chapter 1, L. leucocephala seeds were ground and the resulting flour, homogenized in 100 mM sodium phosphate buffer, pH 7. The suspension obtained was centrifuged (15,000 x g at 4 ° C for 30 min). After centrifugation, the obtained supernatant was denominated: Total extract (TE), cotyledon extract (CE) and shell extract (SE). The protein content, proteolytic, protease inhibitory, chitinolytic and anthelmintic activity of L. leucocephala extracts on H. contortus were verified. For each treatment, the effective concentration (EC) of the samples on the nematode was calculated. The extracts TE and CE presented a high protein content, besides having proteolytic, chitinolytic and protease inhibitory activity and inhibiting egg hatching (EC50 0.33 and 0.48 mg mL -1, respectively). However, L. leucocephala samples did not inhibit the larvae of H. contortus. It is concluded that proteins are correlated with the action of L. leucocephala on H. contortus. In Chapter 2, the parts of S. anthelmia were separated into leaves, roots and stem and, washed with distilled water, lyophilized and subsequently crushed. Subsequently the proteins were solubilized in 100 mM sodium phosphate buffer, pH 7. The suspension was centrifuged (15,000 x g at 4 ° C for 30 min) and the supernatant collected for analysis. From the ammonium sulfate precipitation (0-90%) of S. anthelmia extracts and membrane dialysis with 14 kDa molecular exclusion, protein fractions were obtained and denominated: Leaf protein fraction (LPF), stem protein fraction (SPF), root protein fraction (RPF). The samples were submitted to analysis by high performance liquid chromatography for the detection of compounds of secondary metabolism and mass spectrometry to identify possible bioactive proteins present. The protein content, proteolytic, protease inhibitory, chitinolytic, haemagglutinating and anthelmintic action of S. anthelmia protein fractions on H. contortus were verified. For each treatment, the effective concentration (EC) of the samples on the nematode was calculated. Fractions of S. anthelmia had an inhibitory effect on egg hatching, and this effect was more pronounced by LPF (EC50 0.17 mg mL -1). In addition, greater inhibition of larvae by LPF and RPF (EC50 0.27 and 0.25 mg mL -1, respectively) was observed. The protein fractions of root, stem and leaf of S. anthelmia were effective on the inhibition of larval migration (EC50 0.11, 0.14 and 0.21 mg mL -1, respectively). No secondary metabolite compounds were detected in the S. anthelmia fractions, while several proteins with anthelmintic potential were identified by mass spectrometry. It is concluded that proteins are correlated with the action of S. anthelmia on H. contortus, having potential for the development of anthelmintic products. Thus, the bioactive proteins present in L. leucocephala and S. anthelmia have promising pharmacological properties for the control of the H. contortus nematode.