Livio Martins Costa Junior

An ethnopharmacological assessment of the use of plants against parasitic diseases in humans and animals.

ETHNOPHARMACOLOGICAL RELEVANCE Ethnobotanical surveys are detecting an increasing frequency of exotic plant species in pharmacopeias, which has led researchers to investigate the role of such species in traditional medical systems. According to the diversification hypothesis, exotic species are included to complete pharmacopeias, i.e., to treat diseases for which no native species are known, thus broadening the scope of the plant repertoire. MATERIALS AND METHODS The present study was conducted at two rural communities in northeastern Brazil aiming at a survey of the plants known or used by the population to treat endo- and ectoparasitic diseases in humans and animals. In addition, plant extracts exhibiting acaricide activity were assessed using the engorged female immersion and larval packet tests (LPT). RESULTS The results of the present study showed a tendency for native species to be used against ectoparasites and exhibit a broader scope of use compared to exotic species. In turn, exotic species were predominantly indicated to treat diseases caused by endoparasites, although there was an overlap of native and exotic species relative to some therapeutic purpose, e.g., ticks. Only two of the plant species tested exhibited acaricide activity (Nicotiana glauca Graham and Croton blanchetianus Baill.), and in both cases, the activity was weak. CONCLUSION The ethnobotanical data do not fully support the suggested hypothesis. Overall, the wide versatility of exotic species was not exclusively used to treat parasitic diseases in humans and animals. In addition, the selection of acaricide plants based on the ethnopharmacological study generated uninteresting results.

Standardization and application of the tetraprimer ARMS-PCR technique for screening of the E198A SNP in the β-tubulin gene of hookworm populations in Brazil

The tetraprimer ARMS-PCR technique is efficient for SNP detection and can be used to search for polymorphisms associated with drug resistance. However, the establishment of this methodology is not always straightforward because of the constraints on primer design due to the restrictions of the polymorphic regions. Here, we describe the standardization of the tetraprimer ARMS-PCR methodology for the detection of a SNP at codon 198 of the Ancylostoma caninum β-tubulin gene. This SNP is associated with resistance to albendazole in various nematodes. The methodology was used to screen 327 individuals from 6 different locations. No mutation was found in any of the samples. This methodology will be useful for screening for the E198A SNP in the β-tubulin gene of canine hookworms in a broader population to determine whether this SNP is associated with benzimidazole resistance in this species. The method could also be adapted for the analysis of other SNPs in other nematode species.

Chenopodium ambrosioides L. Reduces Synovial Inflammation and Pain in Experimental Osteoarthritis

The chronicity of osteoarthritis (OA), characterized by pain and inflammation in the joints, is linked to a glutamate receptor, N-methyl-D-aspartate (NMDA). The use of plant species such as Chenopodium ambrosioides L. (Amaranthaceae) as NMDA antagonists offers a promising perspective. This work aims to analyze the antinociceptive and anti-inflammatory responses of the crude hydroalcoholic extract (HCE) of C. ambrosioides leaves in an experimental OA model. Wistar rats were separated into six groups (n = 24): clean (C), negative control (CTL-), positive control (CTL+), HCE0.5, HCE5 and HCE50. The first group received no intervention. The other groups received an intra-articular injection of sodium monoiodoacetate (MIA) (8 mg/kg) on day 0. After six hours, they were orally treated with saline, Maxicam plus (meloxicam + chondroitin sulfate) and HCE at doses of 0.5 mg/kg, 5 mg/kg and 50 mg/kg, respectively. After three, seven and ten days, clinical evaluations were performed (knee diameter, mechanical allodynia, mechanical hyperalgesia and motor activity). On the tenth day, after euthanasia, synovial fluid and draining lymph node were collected for cellular quantification, and cartilage was collected for histopathological analysis. Finally, molecular docking was performed to evaluate the compatibility of ascaridole, a monoterpene found in HCE, with the NMDA receptor. After the third day, HCE reduced knee edema. HCE5 showed less cellular infiltrate in the cartilage and synovium and lower intensities of allodynia from the third day and of hyperalgesia from the seventh day up to the last treatment day. The HCE5 and HCE50 groups improved in forced walking. In relation to molecular docking, ascaridole showed NMDA receptor binding affinity. C. ambrosioides HCE was effective in the treatment of OA because it reduced synovial inflammation and behavioral changes due to pain. This effect may be related to the antagonistic effect of ascaridole on the NMDA receptor.

Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus

The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts.

Anthelmintic activity of plant extracts from Brazilian savanna

Helminth infections represent a serious problem for the production of small ruminants that is currently aggravated by resistance to anthelmintic products and has induced a search for control alternatives, such as natural products. In this study, extracts of Turnera ulmifolia L. (leaves and roots), Parkia platycephala Benth. (leaves and seeds) and Dimorphandra gardneriana Tul. (leaves and bark), which have been cited in ethnoveterinary studies and selected naturally by goats in the cerrado (Brazilian savanna), were tested in vitro against Haemonchus contortus. Hydroacetonic (ACT) and hydroalcoholic (ETH) extracts were evaluated using an Egg Hatching Assay (EHA), a Larval Exsheathment Inhibition Assay (LEIA) and a Larval Development Assay (LDA). A second set of incubations was performed using polyvinylpolypyrrolidone (PVPP) to determine the influence of polyphenols on the anthelmintic effects of EHA and LEIA. Data from each extract were used to calculate inhibition concentrations (IC50). All tested extracts showed activity against at least one life stage of H. contortus. The use of PVPP revealed that the tannins are not the only extracts of secondary metabolites responsible for the anthelmintic effects. The results showed clear in vitro anthelmintic activities against H. contortus at different stages and indicated the potential use of these species as a promising alternative approach to control helminthic infections of small ruminants.

Parasitism by Ixodiphagus Wasps (Hymenoptera: Encyrtidae) in Rhipicephalus sanguineus and Amblyomma Ticks (Acari: Ixodidae) in Three Regions of Brazil

ABSTRACT Hymenopteran parasitoids have been shown to be of potential value in tick biocontrol. Tick parasitoids has been reported only once in Brazil, over 95 yr ago when Ixodiphagus hookeri (Howard) was reported parasitizing Rhipicephalus sanguineus (Latreille) nymphs in Rio de Janeiro. Herein, we report the occurrence of Ixodiphagus spp. in ticks from three different regions of Brazil. In the state of Maranhão, in northeastern Brazil, parasitoids were detected in R. sanguineus nymphs on three occasions, during August 2009 and September 2010 (dry season), and January 2011 (rainy season). All parasitoids found in northeastern Brazil were identified as I. hookeri. In the state of Mato Grosso do Sul (west—central Brazil), one Amblyomma sp. engorged nymph was shown to be parasitized by I. hookeri. In the state of Rondônia (northern Brazil), one Amblyomma sp. engorged nymph was parasitized by Ixodiphagus texanus (Howard). Because Ixodiphagus spp. are present in ecologically distinct and geographically distant areas of Brazil, they are of potential use for biocontrol in the country.

In vitro acaricidal activity of Crescentia cujete L. fruit pulp against Rhipicephalus microplus

Cattle tick (Rhipicephalus microplus) is an important constraint on livestock production, particularly in tropical and subtropical areas. Synthetic acaricides are the primary method of control this thick. Resistance of R. microplus to synthetic acaricides has given rise to the need of new scientific investigations on alternative ways to control it. The objective of this work was to conduct a chemical investigation of Crescentia cujete L. fruit pulp and to evaluate the acaricide effect on R. microplus larvae of extracts, fractions, and isolated substances from this species. Crescentia cujete is a Bignoniaceae and it is characteristic of tropical climates. Its most distinctive feature is its fruit, which is spherical and gourd-like, with a hard woody shell and gelatinous pulp. The fruit pulp of C. cujete was extracted with ethanol by maceration yielding an extract and a residue. The extract was partitioned giving an ethyl acetate phase (EAF) that was fractionated by chromatographic procedures yielding cinnamic acid and benzoic acid. The remained botanic material from the ethanol extraction was further and successively extracted with ethyl ether and methanol under reflux. The acaricide effect of all extracts and fractions was evaluated against R. microplus using the larval packet test. The best result was observed for the EAF that caused 100% of mortality when a 10% solution was used and with LC50 of 5.9%. Cinnamic acid was also tested showing 63.0% of mortality with LC50 of 6.6% at the same concentrations and time of the experiment. The data obtained in this study provides information related to the chemical nature and the acaricide activity of the C. cujete epicarp that has not been previously reported. This study shows that the ethyl acetate phase of the ethanol extract of C. cujete fruit pulp is a potential alternative control for R. microplus and its activity is in part associated to cinnamic acid, its major compound.