Nitazoxanide Inhibits the Bifunctional Enzyme GlG6PD::6PGL of Giardia lamblia: Biochemical and In Silico Characterization of a New Druggable Target.

Giardiasis, which is caused by Giardia lamblia infection, is a relevant cause of morbidity and mortality worldwide. Because no vaccines are currently available to treat giardiasis, chemotherapeutic drugs are the main options for controlling infection. Evidence has shown that the nitro drug nitazoxanide (NTZ) is a commonly prescribed treatment for giardiasis; however, the mechanisms underlying NTZ's antigiardial activity are not well-understood. Herein, we identified the glucose-6-phosphate::6-phosphogluconate dehydrogenase (GlG6PD::6PGL) fused enzyme as a nitazoxanide target, as NTZ behaves as a GlG6PD::6PGL catalytic inhibitor. Furthermore, fluorescence assays suggest alterations in the stability of GlG6PD::6PGL protein, whereas the results indicate a loss of catalytic activity due to conformational and folding changes. Molecular docking and dynamic simulation studies suggest a model of NTZ binding on the active site of the G6PD domain and near the structural NADP+ binding site. The findings of this study provide a novel mechanistic basis and strategy for the antigiardial activity of the NTZ drug.

Pyridyl Methylsulfinyl Benzimidazole Derivatives as Promising Agents against Giardia lamblia and Trichomonas vaginalis.

Protozoan parasites, such as Giardia lamblia and Trichomonas vaginalis, cause the most prevalent infections in humans in developing countries and provoke significant morbidity and mortality in endemic countries. Despite its side-effects, metronidazole is still the drug of choice as a giardiacidal and trichomonacidal tissue-active agent. However, the emergence of metronidazole resistance and its evolved strategies of parasites to evade innate host defenses have hindered the identification and development of new therapeutic strategies against these parasites. Here, we tested five synthesized benzimidazole derivatives as possible drugs for treating giardiasis and trichomoniasis, probing the bifunctional enzyme glucose 6-phosphate dehydrogenase::6-phosphogluconolactone from G. lamblia (GlG6PD::6PGL) and T. vaginalis (TvG6PD::6PGL) as a drug target. The investigated benzimidazole derivatives were H-B2M1, H-B2M2, H2N-BZM6, O2N-BZM7, and O2N-BZM9. The recombinant enzymes were used in inhibition assays, and in silico computational predictions and spectroscopic studies were applied to follow the structural alteration of the enzymes and identify the possible mechanism of inhibition. We identified two potent benzimidazole compounds (O2N-BZM7 and O2N-BZM9), which are capable of inhibiting both protozoan G6PD::6PGL enzymes and in vitro assays with these parasites, showing that these compounds also affect their viability. These results demonstrate that other therapeutic targets of the compounds are the enzymes GlG6PD::6PGL and TvG6PD::6PGL, which contribute to their antiparasitic effect and their possible use in antigiardial and trichomonacidal therapies.

Giardia lamblia G6PD::6PGL Fused Protein Inhibitors Decrease Trophozoite Viability: A New Alternative against Giardiasis.

Treatments to combat giardiasis have been reported to have several drawbacks, partly due to the drug resistance and toxicity of current antiparasitic agents. These constraints have prompted many researchers to investigate new drugs that act against protozoan parasites. Enzyme inhibition is an important means of regulating pathogen metabolism and has recently been identified as a significant alternative target in the search for new treatments. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase (G6PD::6PGL) is a bifunctional enzyme involved in the pentose phosphate pathway (PPP) in Giardia lamblia (G. lamblia). The G. lamblia enzyme is unusual since, unlike the human enzyme, it is a fused enzyme. Here, we show, through inhibition assays, that an in-house chemical library of 120 compounds and four target compounds, named CNZ-7, CNZ-8, CMC-1, and FLP-2, are potent inhibitors of the G. lamblia G6PD::6PGL fused enzyme. With a constant (k2) of 2.3, 3.2, and 2.8 M−1 s−1, respectively, they provoke alterations in the secondary and tertiary protein structure and global stability. As a novel approach, target compounds show antigiardial activity, with IC50 values of 8.7, 15.2, 15.3, and 24.1 µM in trophozoites from G. lamblia. Moreover, these compounds show selectivity against G. lamblia, since, through counter-screening in Caco-2 and HT29 human cells, they were found to have low toxicity. This finding positions these compounds as a potential and attractive starting point for new antigiardial drugs.

Discovery of Benzopyrrolizidines as Promising Antigiardiasic Agents.

Current treatments for giardiasis include drugs with undesirable side effects, which increase the levels of therapeutic desertion and promote drug resistance in the parasites. Herein, we describe the antigiardiasic evaluation on Giardia lamblia trophozoites of a structurally diverse collection of 74 molecules. Among these scaffolds, we discovered a benzopyrrolizidine derivative with higher antigiardiasic activity (IC50 = 11 µM) and lower cytotoxicity in human cell cultures (IC50 = 130 µM) than those displayed by the current gold-standard drugs (metronidazole and tinidazole). Furthermore, this compound produced morphologic modifications of trophozoites, with occasional loss of one of the nuclei, among other changes not observed with standard giardicidal drugs, suggesting that it might act through a novel mechanism of action.

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability.

This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis (T. vaginalis). The glucose-6-phosphate dehydrogenase (g6pd) gene from T. vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tvg6pd::6pgl gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP+) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP+ were performed. These results revealed that the protein becomes more stable in the presence of the NADP+. In addition, we determined the dissociation constant for the binding (Kd) of NADP+ in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused.

Antigiardiasic activity of Cu(II) coordination compounds: Redox imbalance and membrane damage after a short exposure time.

Giardiasis is a widespread illness that affects inhabitants of underdeveloped countries, being children and seniors the highest risk population. The several adverse effects produced by current therapies besides its increasing ineffectiveness due to the appearance of resistant strains evidence the urgent need for new therapeutic approaches. We present the antigiardiasic effect of eight Cu(II) coordination compounds, which belong to the family Casiopeínas. Two of them, 4,7-diphenyl-1,10-phenanthroline(acetylacetonato)copper(II) nitrate (CasIII-Ha,36 µM) and 4,7-diphenyl-1,10-phenanthroline(glycinato)copper(II) nitrate (CasI-gly,36 µM) have shown the best antiproliferative effect in Giardia intestinalis trophozoite cultures, both with the higher lipophilic character of the series. The antiproliferative effect of these coordination compounds is attributable to its capacity to interact with the cellular membrane and to increase reactive oxygen species (ROS) concentration within the parasite since the first hours of exposure, (2-6 h). We found that these compounds mainly induced the cell death of trophozoites by apoptosis, contrary to metronidazole, which induces apoptosis and necrosis in the same ratio. The cytotoxic effects on lymphocytes and macrophages isolated from human peripheral blood allowed us to establish a selectivity index and in turn, identify and propose the best candidates to continue with the assays in animal models. The selected molecules do not include the most active compounds against trophozoites, instead of that, we propose the compounds 4',4'-dimethyl-2,2'-bipyridine(acetylacetonato)copper(II) nitrate (CasIII-ia,IC50 = 156 µM) and 4,7-dimethyl-1,10-phenanthroline(acetylacetonato) copper(II) nitrate (CasIII-Ea,IC50 = 125 µM), which possess an antiproliferative efficacy comparable with Metronidazole but also are those that produce the lowest effect on the viability of human lymphocytes and macrophages.

Identification of the NADP+ Structural Binding Site and Coenzyme Effect on the Fused G6PD::6PGL Protein from Giardia lamblia.

Giardia lambia is a flagellated protozoan parasite that lives in the small intestine and is the causal agent of giardiasis. It has been reported that G. lamblia exhibits glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in the pentose phosphate pathway (PPP). Our group work demonstrated that the g6pd and 6pgl genes are present in the open frame that gives rise to the fused G6PD::6PGL protein; where the G6PD region is similar to the 3D structure of G6PD in Homo sapiens. The objective of the present work was to show the presence of the structural NADP+ binding site on the fused G6PD::6PGL protein and evaluate the effect of the NADP+ molecule on protein stability using biochemical and computational analysis. A protective effect was observed on the thermal inactivation, thermal stability, and trypsin digestions assays when the protein was incubated with NADP+. By molecular docking, we determined the possible structural-NADP+ binding site, which is located between the Rossmann fold of G6PD and 6PGL. Finally, molecular dynamic (MD) simulation was used to test the stability of this complex; it was determined that the presence of both NADP+ structural and cofactor increased the stability of the enzyme, which is in agreement with our experimental results.

Biochemical Characterization and Structural Modeling of Fused Glucose-6-Phosphate Dehydrogenase-Phosphogluconolactonase from Giardia lamblia.

Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway and is highly relevant in the metabolism of Giardialamblia. Previous reports suggested that the G6PD gene is fused with the 6-phosphogluconolactonase (6PGL) gene (6pgl). Therefore, in this work, we decided to characterize the fused G6PD-6PGL protein in Giardialamblia. First, the gene of g6pd fused with the 6pgl gene (6gpd::6pgl) was isolated from trophozoites of Giardialamblia and the corresponding G6PD::6PGL protein was overexpressed and purified in Escherichia coli. Then, we characterized the native oligomeric state of the G6PD::6PGL protein in solution and we found a catalytic dimer with an optimum pH of 8.75. Furthermore, we determined the steady-state kinetic parameters for the G6PD domain and measured the thermal stability of the protein in both the presence and absence of guanidine hydrochloride (Gdn-HCl) and observed that the G6PD::6PGL protein showed alterations in the stability, secondary structure, and tertiary structure in the presence of Gdn-HCl. Finally, computer modeling studies revealed unique structural and functional features, which clearly established the differences between G6PD::6PGL protein from G. lamblia and the human G6PD enzyme, proving that the model can be used for the design of new drugs with antigiardiasic activity. These results broaden the perspective for future studies of the function of the protein and its effect on the metabolism of this parasite as a potential pharmacological target.

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom.

BACKGROUND: Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. METHODS: The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. RESULTS: P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. CONCLUSION: These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom

Background Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. Methods The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. Results P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. Conclusion These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.

Hemolytic, anticancer and antigiardial activity of Palythoa caribaeorum venom

Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. Methods: The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. Results: P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. Conclusion: These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.(AU)

Disulfiram as a novel inactivator of Giardia lamblia triosephosphate isomerase with antigiardial potential.

Giardiasis, the infestation of the intestinal tract by Giardia lamblia, is one of the most prevalent parasitosis worldwide. Even though effective therapies exist for it, the problems associated with its use indicate that new therapeutic options are needed. It has been shown that disulfiram eradicates trophozoites in vitro and is effective in vivo in a murine model of giardiasis; disulfiram inactivation of carbamate kinase by chemical modification of an active site cysteine has been proposed as the drug mechanism of action. The triosephosphate isomerase from G. lamblia (GlTIM) has been proposed as a plausible target for the development of novel antigiardial pharmacotherapies, and chemical modification of its cysteine 222 (C222) by thiol-reactive compounds is evidenced to inactivate the enzyme. Since disulfiram is a cysteine modifying agent and GlTIM can be inactivated by modification of C222, in this work we tested the effect of disulfiram over the recombinant and trophozoite-endogenous GlTIM. The results show that disulfiram inactivates GlTIM by modification of its C222. The inactivation is species-specific since disulfiram does not affect the human homologue enzyme. Disulfiram inactivation induces only minor conformational changes in the enzyme, but substantially decreases its stability. Recombinant and endogenous GlTIM inactivates similarly, indicating that the recombinant protein resembles the natural enzyme. Disulfiram induces loss of trophozoites viability and inactivation of intracellular GlTIM at similar rates, suggesting that both processes may be related. It is plausible that the giardicidal effect of disulfiram involves the inactivation of more than a single enzyme, thus increasing its potential for repurposing it as an antigiardial drug.

RNAi-Mediated Specific Gene Silencing as a Tool for the Discovery of New Drug Targets in Giardia lamblia; Evaluation Using the NADH Oxidase Gene.

The microaerophilic protozoan Giardia lamblia is the agent causing giardiasis, an intestinal parasitosis of worldwide distribution. Different pharmacotherapies have been employed against giardiasis; however, side effects in the host and reports of drug resistant strains generate the need to develop new strategies that identify novel biological targets for drug design. To support this requirement, we have designed and evaluated a vector containing a cassette for the synthesis of double-stranded RNA (dsRNA), which can silence expression of a target gene through the RNA interference (RNAi) pathway. Small silencing RNAs were detected and quantified in transformants expressing dsRNA by a stem-loop RT-qPCR approach. The results showed that, in transformants expressing dsRNA of 100-200 base pairs, the level of NADHox mRNA was reduced by around 30%, concomitant with a decrease in enzyme activity and a reduction in the number of trophozoites with respect to the wild type strain, indicating that NADHox is indeed an important enzyme for Giardia viability. These results suggest that it is possible to induce the G. lamblia RNAi machinery for attenuating the expression of genes encoding proteins of interest. We propose that our silencing strategy can be used to identify new potential drug targets, knocking down genes encoding different structural proteins and enzymes from a wide variety of metabolic pathways.

Toxocara canis: Analysis of the kinetics of antigen release and antibody production in an in vivo model for the detection of past or present infection.

Worldwide, Toxocara canis is an important zoonotic nematode of public health concern. This soil-transmitted helminth causes visceral larva and ocular larva migrans in paratenic hosts. The detection of T. canis larva migrans is complicated because current immunological tests detect only IgG antibodies, which can cross-react with antigens from other parasites and cannot distinguish between the past and present infection. Analysis of antigen release and antibody production could help improve the detection of larva migrans. Here, we report the kinetics of antigen release, IgM and IgG production in an in vivo model for the detection of past or present infection. We used four groups of seven mice: two groups infected orally with 50 or 100 embryonated eggs, and the other two infected intraperitoneally with 50 or 100 live larvae. We obtained blood samples at 0, 3, 7, and 14days and, then, every two weeks until day 140. Sandwich ELISA and indirect ELISA were performed for antigen capture and the detection of immunoglobulins, respectively. Mice inoculated with larvae developed an immune response faster than those inoculated with eggs. In all groups, antigen capture was positive starting at 3days until 140days post-inoculation (dpi). Detection of immunoglobulins was at 14 or 28dpi in mice inoculated with larvae or eggs, respectively. Negative IgM values were detected at days 98 and 112. The samples remained positive for IgG until the last day of the experiment. Data suggest that in mice inoculated with T canis eggs, some larvae did not hatch, others died or never reached the bloodstream. Based on our model, we propose that there is early infection when only antigens are present, and active larva migrans when antigen and immunoglobulins are detected, implying an immune response of the host against the antigen. Our study offers a view into the parasite-host relationship and enables us to infer if there are live larvae. Additionally, these findings provide a foundation for the diagnosis and differentiation of recent infection and active larva migrans.

Piqueria trinervia as a source of metabolites against Giardia intestinalis.

CONTEXT: Piqueria trinervia Cav. (Asteraceae) is a plant species with a long history in traditional medicine to cure diarrhoea and other digestive disorders. OBJECTIVE: The study investigates the antigiardial activity of piquerol, trinervinol, red oil and two fractions (F1 and F2) from P. trinervia. MATERIALS AND METHODS: P. trinervia was collected in the Ajusco in Mexico City. Aerial parts were ground and mixed with water to obtain the extract, which was treated with dichloromethane to isolate piquerol and trinervinol (P & T). Remnants were the red oil, fractions 1 and 2 (RO, F1 & F2). Trophozoites of Giardia intestinalis were treated with P, T, RO, F1 and F2 at different concentrations (0.78-200 µg/mL) for 48 h. Antigiardial activity was measured using the methylene blue reduction, and the cytotoxicity assayed on human fibroblasts and Vero cells by reduction of tetrazolium salts. RESULTS: Trinervinol and piquerol showed antigiardial activity with an IC50 = 2.03 and 2.42 µg/mL, and IC90 = 13.03 and 8.74 µg/mL, respectively. The concentrations of trinervinol (CC50 = 590 µg/mL) and piquerol (CC50 = 501 µg/mL) were not cytotoxic to human fibroblasts. CONCLUSIONS: Compounds from P. trinervia showed antigiardial activity; to enhance this activity, piquerol and trinervinol can be chemically modified.

In vitro and in vivo cysticidal activity of extracts and isolated flavanone from the bark of Prunus serotina: A bio-guided study.

Currently, neurocysticercosis treatment involves two drugs: albendazole and praziquantel; however, their efficacy is suboptimal and new cysticidal drugs are needed. The present paper reports the cysticidal activity of extracts of the bark from Prunus serotina against Taenia crassiceps cysts and the isolation and identification of the main components of the most active extract. Results showed that all extracts displayed in vitro cysticidal activity (EC50=17.9-88.5µg/mL), being the methanolic the most active and selective. Also, methanolic extract exhibited in vivo efficacy at 300mg/kg which was similar to that obtained with albendazole. Bio-guided fractionation of methanolic extract led the isolation of 2,3-dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one (naringenin, NGN), 3,4,5-trimethoxybenzoic acid and 1,3,5-trimethoxybenzene. NGN exhibited in vitro activity, in a time-concentration-dependent manner (EC50=89.3µM]. Furthermore, NGN at a dose of 376.1µmol/kg displayed similar in vivo efficacy than those obtained with albendazole at 188.4µmol/kg. NGN also caused a high level of damage in all parasite tissue in a similar manner than that observed with the methanolic extract. This study represents the first report of the cysticidal properties of the bark of P. serotina. NGN was identified as the main active compound of this specie and other studies are required to explore the potential of this flavanone as cysticidal agent.

Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia.

Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect-in a rapid, sensitive, specific, and reproducible way-the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi) pathway in G. lamblia.

In vitro activity of 'Mexican Arnica' Heterotheca inuloides Cass natural products and some derivatives against Giardia intestinalis.

Giardiasis is a gastrointestinal disease that affects humans and other animals caused by parasitic protists of the genus Giardia. Giardia intestinalis (Syn. Giardia lamblia; Giardia duodenalis) infections can cause acute or chronic diarrhoea, dehydration, abdominal discomfort and weight loss. Metronidazole is the most widely used drug for treating giardiasis. Although effective, metronidazol has undesirable secondary effects. Plants used in traditional medicine as antidiarrhoeals or antiparasitics may represent alternative sources for new compounds to treat giardiasis. Heterotheca inuloides Cass. (Asteraceae/Compositae) plant is widely used in Mexican traditional medicine. The following secondary metabolites were isolated from H. inuloides flowers: 7-hydroxy-3,4-dihydrocadalene (1), 7-hydroxycadalene (2), 3,7-dihydroxy-3(4H)-isocadalen-4-one (3), 1R,4R-hydroxy-1,2,3,4-tetrahydrocadalen-15-oic acid (4), quercetin (5), quercetin-3,7,3'-trimethyl ether (6), quercetin-3,7,3',4'-tetramethyl ether (7) and eriodictyol-7,4'-dimethyl ether (8). The activity of these compounds against Giardia intestinalis trophozoites was assessed in vitro as was the activity of the semisynthetic compounds 7-acetoxy-3,4-dihydrocadalene (9), 7-benzoxy-3,4-dihydrocadalene (10), 7-acetoxycadalene (11), 7-benzoxycadalene (12), quercetin pentaacetate (13) and 7-hydroxycalamenene (14). Among these, 7-hydroxy-3,4-dihydrocadalene (1) and 7-hydroxycalamenene (14) were the most active, whereas the remaining compounds showed moderate or no activity. The G. intestinalis trophozoites exposed to compound 1 showed marked changes in cellular architecture along with ultrastructural disorganization. The aim of this study was to evaluate the giardicidal activity of selected H. inuloides metabolites and some semisynthetic derivatives using an in vitro experimental model of giardiasis.

In vitro activity of the F-6 fraction of oregano against Giardia intestinalis.

Giardiosis is a neglected parasitic disease that produces diarrhoea and different degrees of malabsorption in humans and animals. Its treatment is based on derivatives of 5-nitroimidazoles, benzimidazoles, nitrofuranes, acridine and nitrotiazoles. These drugs produce undesirable secondary effects, ranging from a metallic taste in the mouth to genetic damage and the selection of resistant strains; therefore, it is necessary to develop new therapeutic alternatives. We demonstrated that a 2-h treatment with 2·87 µg ml(-1) of fraction 6 of Lippia graveolens (F-6) was sufficient to kill half of an experimental Giardia intestinalis (Syn. G. duodenalis, G. lamblia) population, based on the reduction of MTT-tetrazolium salt levels. F-6 breaks the nuclear envelope and injures the ventral suckling disc. The major compounds of F-6 were characterized as naringenin, thymol, pinocembrin and traces of compounds not yet identified. The results suggest that Lippia is a potential source to obtain compounds with anti-Giardia activity. This knowledge is an important starting point to develop new anti-giardial drugs. Future studies will be required to establish the efficacy of F-6 in vivo using an animal model.

Oregano (Lippia spp.) kills Giardia intestinalis trophozoites in vitro: antigiardiasic activity and ultrastructural damage.

In the world, giardiosis is still a very important parasitic disease; only in Asia, Africa and America, there are more than 200 million of infected people in a year. The usual treatments are drugs that produce undesirable secondary effects, perhaps favouring the resistant strain selection. One alternative is to research compounds from plants used as antidiarrhoeic or antiparasitic in the traditional medicine. In a previous work, we found that Lippia beriandieri (Oregano) revealed to be more potent than tinidazole, a common antigiardiasic drug. In this current work, we tested the cell viability by re-culture and reduction of MTT-tetrazolium salts to MTT-formazan, and we showed the effect of oregano ethanolic extracts against Giardia intestinalis (synonyms: Giardia duodenalis, Giardia lamblia) trophozoites at concentrations ranging form 58 to 588 microg. We demonstrated the ultrastructural injury produced by oregano extracts in this parasite. Trophozoites lost their size and shape and showed damage in nucleus structure, perhaps by breaking the pattern of nucleoskeleton proteins.