Trypanocidal constituents in plants 5. Evaluation of some Mexican plants for their trypanocidal activity and active constituents in the seeds of Persea americana.

Crude extracts of Mexican medicinal plants were screened for trypanocidal activity against Trypanosoma cruzi, which is the etiological agent for Chagas' disease, one of the most serious protozoan diseases in Latin America. There were 71 kinds of methanolic and other organic extracts from 65 plants, which were newly examined by a preliminary screening test to observe immobilization of epimastigotes and trypomastigotes of T. cruzi in vitro. The MeOH extract of seeds of Persea americana (avocado) showed moderate activity against epimastigotes. In order to identify the principal compounds for the activity, the MeOH extract was subjected to bioassay-guided fractionation. From the active fractions, six 1,2,4-trihydroxyheptadecane derivatives and two 1,2,4-trihydroxynonadecane derivatives including a new one were isolated. These compounds showed moderate activity against epimastigotes and trypomastigotes.

Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects.

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb-e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb-e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb-e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.

Trypanocidal constituents in plants 4. Withanolides from the aerial parts of Physalis angulata.

The constituents of the aerial parts of Physalis angulata (Solanaceae) were investigated based on the plant's trypanocidal activity against epimastigotes of Trypanosoma cruzi, the etiologic agent for Chagas' disease. Four new withanolides were isolated, along with six known ones, from the active fraction. Their structures were determined by spectroscopic analysis. Trypanocidal activity against trypomastigotes, an infectious form of T. cruzi, was also estimated, as well as cytotoxic activity against human uterine carcinoma (HeLa) cells in vitro. Evaluation of trypanocidal activity using the colorimetric reagent Cell Counting Kit-8 was also examined.

Trypanocidal constituents in plants 3. Leaves of Garcinia intermedia and heartwood of Calophyllum brasiliense.

The constituents of the leaves of Garcinia intermedia and heartwood of Calophyllum brasiliense were investigated based on their trypanocidal activity against epimastigotes of Trypanosoma cruzi, the etiologic agent of Chagas' disease. As the active components, the polyisoprenylated benzophenone derivative guttiferone A and the xanthone 8-desoxygartanin were isolated along with the biflavonoids podocarpusflavone A and amentoflavone, and friedelin from the former. Three xanthones, jacareubin, 6-deoxyjacareubin, and 1,3,5,6-tetrahydroxy-2-(3-methyl-2-butenyl)xanthone from the latter showed activity. The trypanocidal activity of these compounds against trypomastigotes, an infectious form of T. cruzi, was examined as well as gossypol, berberine chloride, and harmine for comparison.

Trypanocidal constituents in plants 2.xanthones from the stem bark of Garcinia subelliptica.

The constituents of the stem bark of Garcinia subelliptica (Guttiferae) were investigated based on its trypanocidal activity against epimastigotes of Trypanosoma cruzi, the etiologic agent for Chagas' disease. As the active components, nine xanthones were isolated including two new ones, 4-hydroxybrasilixanthone B and 1,3,5,6-tetrahydroxy-4,7,8-tri(3-methyl-2-butenyl)xanthone. Their structures were determined by spectroscopic analysis. Trypanocidal activity against trypomastigotes, an infectious form of T. cruzi, was also estimated as well as cytotoxic activity. Fukugetin, the major component of the bark, showed no activity.

Ursolic acid as a trypanocidal constituent in rosemary.

The MeOH extract of the leaves of rosemary (Rosmarinus officinalis L.) completely inhibited the motility of cultured epimastigotes of Trypanosoma cruzi at the concentration of 2 mg/ml after 2 h of incubation. Activity-guided fractionation of the MeOH extract has resulted in the isolation of three triterpene acids, betulinic, oleanolic and ursolic acids. Ursolic acid stopped the movement of all T. cruzi epimastigotes at the minimum concentration (MC(100)) of 40 micro g/ml (88 micro M) after 48 h of incubation. Oleanolic acid was less active (MC(100): 250 micro g/ml, 550 micro M) and betulinic acid was practically inactive.

Trypanocidal constituents in plants 1. Evaluation of some Mexican plants for their trypanocidal activity and active constituents in Guaco, roots of Aristolochia taliscana.

Crude extracts of Mexican medicinal plants were screened for trypanocidal activity against Trypanosoma cruzi, which is the etiological agent for Chagas' disease, one of the most serious protozoan diseases in Latin America. There were 43 kinds of methanolic and other organic extracts from 39 plants which were examined by the preliminary screening test to see immobilization of epimastigotes of T. cruzi in vitro. Eighteen of them showed activity at the concentration of 2 mg/ml after incubation for 2 h, while 13 showed activity at the concentration of 1 mg/ml after incubation for 48 h. Among them, the MeOH extract of roots of Aristolochia taliscana (Aristolochiaceae), locally known as "Guaco," immobilized all the epimastigotes even at lower concentration of 0.5 mg/ml (48 h). In order to identify principal compounds for this activity, the MeOH extract of Guaco was subjected to bioassay-guided fractionation. From the active fractions, four neolignans, eupomatenoid-7 (1), licarin A (2), eupomatenoid-1 (5) and licarin B (6), and two lignans, austrobailignan-7 (3) and fragransin E1 (4) were isolated. Compounds 1-4 immobilized all the epimastigotes at the minimum concentration of 25-75 microg/ml after incubation for 48 h, while compounds 5 and 6 were inactive. Corresponding concentration of gossypol, berberine chloride and harmine was 280 microg/ml, 300 microg/ml and >500 microg/ml, respectively.

Study of the ethiological agent of gnathostomosis in Nayarit, Mexico.

In order to clarify the specific identity of the etiological agent of human gnathostomosis in Nayarit State, Mexico, morphological and molecular studies were conducted on advanced third stage larvae obtained from human and fish tissue. Cathorops fuerthii from Agua Brava lagoons complex, was the only fish species found to be infected among four species surveyed. Morphological variability does not allow specific identification of the larvae. Internal transcribed spacer 2 of the ribosomal DNA was sequenced for six larvae (five from fish, one from human tissue). Low divergence in the sequences of Nayarit larvae and Gnathostoma binucleatum (0.24% or less) indicate that the larvae examined belong to this species.

Morphology of Gnathostoma spp. isolated from natural hosts in Sinaloa, Mexico.

Gnathostomosis is an emerging public health problem in Sinaloa, Mexico, where an increasing number of human cases have been diagnosed since 1989. The present study was carried out to determine the presence of the parasite in other natural hosts from the area. Birds, fish, opossums and raccoons were captured from local dams and lagoons. The flesh from bird and fish specimens was ground and examined under a 100 W light bulb. Larvae were processed for light and electron microscopy. A total of 368 advanced stage 3 (AL3) larvae were found in 300 ichthyophagous birds, with Egretta alba exhibiting the highest infection rate. A total of 4,156 fish were examined, of which six species were infected with AL3 larvae: Arius guatemalensis (blue sea catfish), Dormitator latifrons (Pacific fat sleeper), Gobiomorus sp. (fat sleeper), Oreochromis sp. (Nile tilapia), Cichlasoma beani (Sinaloan cichlid or green guapote) and Eleotris picta (spotted sleeper). Twenty larvae from birds were used to infect domestic cats and dogs. Young adult worms were recovered from the stomach of a cat with a 17 day infection and from a dog with a 35 day infection. Larvae exhibited four rows of hooklets on the head bulb, whereas the young adults had nine rows of hooklets. The cuticular spines of adult worms along the body evolved from single-pointed, bi- or trifurcated spines. Nuclei were counted in intestinal cells examined in serial sections of larvae recovered from a great heron and a fish, in which a mean of 1.6 nuclei/cell was found, corresponding to data published for Gnathostoma binucleatum. Although the external morphology of both larvae and adults are in agreement with previous descriptions of Gnathostoma spinigerum, the results indicate that natural host infections in Sinaloa may be caused by either G. spinigerum or G. binucleatum.