Auxotrophy to lipoproteins of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> cultivated under axenic conditions

Entamoeba histolytica grows in media without serum but with a mixture of aminoacids, vitamins, lipoproteins, free cholesterol, phospholipids and fatty acids called PACSR. The ability of lipoproteins and free lipids to support growth of three E. histolytica strains (HK9, HMI:IMSS and HM3:IMSS) was analysed. Tubes containing 5 ml culture medium, amino acids, vitamins and either 120–1,200 μg lipoproteins/ml or 0.017–0.10 mg free lipids/ml (predissolved in absolute ethanol) were inoculated with 1 × 10⁴ trophozoites/ml and incubated at 37 °C for 72 h. Amoebae died within 12 h in the presence of any free lipid combination, while those having 240–480 mg lipoproteins/ml reached densities similar to or higher than those of controls (depending on strain). The addition of ethanol (0.1%) to the media produced stable lipid solutions and did not show significant adverse effects. Accordingly, E. histolytica is auxotrophic to lipoproteins and unable to use free cholesterol, phospholipids or fatty acids. Received: 9 November 1999 / Accepted: 9 June 2000     

The in vitro anti-giardial activity of extracts from plants that are used for self-medication by AIDS patients in southern Thailand

This study evaluated the anti-giardial activity of chloroform, methanol and water extracts of 12 medicinal plants (39 extracts), commonly used as self medication by AIDS patients in southern Thailand. The plant extracts and a standard drug, metronidazole, were incubated with 2×10⁵ trophozoites of Giardia intestinalis per millilitre of growth medium in 96-well tissue culture plates under anaerobic conditions for 24 h. The cultures were examined with an inverted microscope and the minimum inhibitory concentration and the IC₅₀ value for each extract was determined. The chloroform extracts from Alpinia galanga, Boesenbergia pandurata, Eclipta prostrata, Piper betle, Piper chaba, Zingiber zerumbet, and the methanol extracts from B. pandurata and E. prostrata were classified as active, i.e. with an IC₅₀ of <100 g/ml, whereas the chloroform extract from Murraya paniculata was classified as being moderately active. This study shows that extracts from some medicinal plants have potential for use as therapeutic agents against G. intestinalis infections.     

The use of microfluorometric method for activity-guided isolation of antiplasmodial compound from plant extracts

In vitro antiplasmodial activity of methanolic extracts of 16 medicinal plants was evaluated by fluorometric assay using PicoGreen. The IC50s, as determined by parasite DNA concentration, ranged from <11 to >200 and <13 to >200 μg/ml for Plasmodium falciparum 3D7 and K1, respectively; and the most active extracts were those from Anogeissus leiocarpus and Terminalia avicennoides (<11–≥14 μg/ml). Aqueous, butanolic, ethyl acetate, and methanolic fractions of these two extracts revealed butanolic fraction to have a relatively better activity (IC₅₀, 10–12 μg/ml). Activity-guided chromatographic separation of the butanolic fraction on Sephadex LH-20 followed by nuclear magnetic resonance and correlation high-performance liquid chromatography revealed the presence of known hydrolysable tannins and some related compounds—castalagin, ellagic acid, flavogallonic acid, punicalagin, terchebulin, and two other fractions. The IC₅₀s of all these compounds ranged between 8–21 μg/ml (8–40 μM) against both the strains. Toxicity assay with mouse fibroblasts showed all the extracts and isolated compounds to have IC₅₀ ≥ 1500 μg/ml, except for Momordica balsamina with <1500 μg/l. All the extracts and isolated compounds did not affect the integrity of human erythrocyte membrane at the observed IC₅₀s. However, adverse effects manifest in a concentration-dependent fashion (from IC₅₀ ≥ 500 μg/ml).     

Antiplasmodial activity of ineupatorolides A from Carpesium rosulatum

Samples of Carpesium genus used as traditional remedies for the treatment of parasite infections were collected, and methanol extracts were obtained by sonication. The ethylacetate-, n-butanol- and H₂O-soluble fractions exhibited weak antiplasmodial activity (IC₅₀ > 100 μg/ml; IC₅₀, 50% inhibitory concentration). However, the chloroform fraction exhibited more impressive antiplasmodial activity (IC₅₀ = 8.2 μg/ml). The antiplasmodial activity of the chloroform fractions was evaluated in vitro against the chloroquine-resistant D10 strain of Plasmodium falciparum. Bioactivity-guided isolation of the chloroform fractions of the whole plants of Carpesium rosulatum has led to the isolation of a sesquiterpene lactone, ineupatorolides A, displaying high antiplasmodial activity (IC₅₀ = 0.007 μg/ml). This is the first report of the isolation of ineupatorolides A from this species and of its remarkable antiplasmodial activity.     

Antiprotozoan activity of Brazilian marine cnidarian extracts and of a modified steroid from the octocoral <Emphasis Type="Italic">Carijoa riisei</Emphasis>

In the present investigation, we have evaluated the antileishmanial and antitrypanosomal activity of methanolic crude extracts obtained from eight species of cnidarians and of a modified steroid isolated from the octocoral Carijoa riisei. The antileishmanial activity of cnidarians crude extracts showed 50% inhibitory concentration (IC₅₀) values in the concentration range between 2.8 and 93.3 μg/mL. Trypomastigotes of Trypanosoma cruzi were less susceptible to the crude extracts, with IC₅₀ values in the concentration range between 40.9 and 117.9 μg/mL. The steroid (18-acetoxipregna-1,4,20-trien-3-one) displayed a strong antileishmanial activity, with an IC₅₀ value of 5.5 μg/mL against promastigotes and 16.88 μg/mL against intracellular amastigotes. The steroid also displayed mammalian cytotoxicity (IC₅₀ of 10.6 μg/mL), but no hemolytic activity was observed at the highest concentration of 12.5 μg/mL. The antileishmanial effect of the steroid in macrophages suggested other mechanism than macrophage activation, as no upregulation of nitric oxide was observed. The antitrypanosomal activity of the steroid resulted in an IC₅₀ value of 50.5 μg/mL. These results indicate the potential of cnidarian natural compounds as antileishmanial drug candidates.     

Effect of oregano (<Emphasis Type="Italic">Origanum vulgare</Emphasis> L.) and thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> L.) essential oils on <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> (Protozoa: Kinetoplastida) growth and ultrastructure

In the present work, we have investigated the effect of essential oils obtained from Origanum vulgare L. (oregano) and Thymus vulgaris L. (thyme) on growth and ultrastructure of diverse evolutive forms of Trypanosoma cruzi. Culture epimastigotes and bloodstream trypomastigotes were incubated for 24 h with different concentrations of oregano or thyme essential oils and with thymol (the main constituent of thyme), and the inhibitory concentration (IC)₅₀ was determined by cell counting. Crude extract of oregano essential oil inhibited epimastigote growth (IC₅₀/24 h = 175 μg/ml) and also induced trypomastigote lysis (IC₅₀/24 h = 115 μg/ml). Thyme essential oil presented IC₅₀/24 h values of 77 μg/ml for epimastigotes and 38 μg/ml for trypomastigotes, while treatment with thymol resulted in an IC₅₀/24 h of 62 μg/ml for epimastigotes and 53 μg/ml for trypomastigotes. Scanning electron microscopy of treated cells showed few morphological alterations at the plasma membrane. Observation by transmission electron microscopy showed cytoplasmic swelling with occasional morphological alterations in plasma and flagellar membrane. Our data indicate that oregano and thyme essential oils are effective against T. cruzi, with higher activity of thyme, and that thymol may be the main component responsible for the trypanocidal activity.     

In vitro antimalarial activity of traditionally used Western Ghats plants from India and their interactions with chloroquine against chloroquine-resistant Plasmodium falciparum

Malaria is a major global public health problem, and the alarming spread of drug resistance and limited number of effective drugs now available underline how important it is to discover new antimalarial compounds. An ethnopharmacological investigation was undertaken on Western Ghats plants traditionally used to treat malaria in India; 50 plants were very carefully selected from a total of 372 plants, and 200 extracts were prepared and tested for in vitro antiplasmodial activity alone and in combination with chloroquine (CQ) against CQ-resistant Plasmodium falciparum (strain MRC-Pf-43). In in vitro antiplasmodial activity, when plant extract alone is used, 29 extracts (or 14.5%) showed significant high in vitro antiplasmodial activity with IC₅₀ values ranging from 3.96 to 4.85 μg/ml, 53 extracts (or 26.5%) showed significant good in vitro antiplasmodial activity with IC₅₀ values ranging from 5.02 to 9.87 μg/ml, and 28 extracts (or 14%) showed significant moderate in vitro antiplasmodial activity with IC₅₀ values ranging from 10.87 to 14 μg/ml, respectively. In combination with CQ, 103 extracts (or 51.5%) showed significant synergistic in vitro antiplasmodial activities with synergistic factor values ranging from 1.03 to 1.92, and these activities were up to a fold higher with CQ, suggesting synergistic interactions of the two drugs. Our investigation has confirmed that above 62.1% of the plant extracts showed moderate to high in vitro antiplasmodial activity when used alone, and in combination with CQ, 55.7% of the extracts showed borderline to good synergistic activity.     

Seaweeds as a source of lead compounds for the development of new antiplasmodial drugs from South East coast of India

The problems of resistant lines of Plasmodium falciparum are escalating. Twelve seaweeds species belong to five different families (Sargassaceae, Gracilariaceae, Hypneaceae, Corallinaceae and Halimedaceae) were collected from Mandapam coastal area, and the seaweeds extracts were tested for in vitro antiplasmodial activity against P. falciparum. Among the tested seaweeds, Gracilaria verrucosa (IC₅₀ 5.55 μg.ml⁻¹) and Hypnea espera (IC₅₀ 8.94 μg.ml⁻¹) showed good antiplasmodial activity, and these results are comparable with positive controls such as artemether (IC₅₀ 4.09 μg.ml⁻¹) and chloroquine (IC₅₀ 19.59 μg.ml⁻¹), respectively. Turbinaria conoides, Sargassum myriocystem, Hypnea valentiae and Jania rubens extracts showed IC₅₀ values between 5 to 50 μg.ml⁻¹. Sargassum sp., Turbinaria decurrens and Halimeda gracilis extracts showed IC₅₀ values between 50 to 100 μg.ml⁻¹. Gracilaria corticata, Jania adherens and Halimeda opuntia extracts showed IC₅₀ value of more than 100 μg.ml⁻¹. Statistical analysis reveals that significant in vitro antiplasmodial activity (P < 0.05) was observed between the concentrations and time of exposure. The chemical injury to erythrocytes was also carried out, and it shows that no morphological changes in erythrocytes by the ethanolic extract of seaweeds extracts after 48 h of incubation. The in vitro antiplasmodial activity might be due to the presence of sugars, proteins, phenols and carboxylic acid in the ethanolic extracts of seaweeds. It is concluded from the present study that the ethanolic extracts of seaweeds of G. verrucosa and Hypnea espera possess lead compounds for development of antiplasmodial drugs.     

In vitro anthelmintic activity of the essential oils of <Emphasis Type="BoldItalic">Zanthoxylum zanthoxyloides</Emphasis> and <Emphasis Type="BoldItalic">Newbouldia laevis</Emphasis> against <Emphasis Type="BoldItalic">Strongyloides ratti</Emphasis>

The need for new anthelmintic with no chemical residues is becoming urgent. In a program aiming at the evaluation of plant as sources of new active molecules, the anthelmintic activities of the essential oils (EOs) obtained from either Zanthoxylum zanthoxyloides seeds or Newbouldia laevis leaves were evaluated against Strongyloides ratti by analyzing the results of two in vitro bioassays. These two plants and their tested parts were retained after an ethnopharmacology survey that confirmed their use by small-scale farmers for treatment of small ruminants affected by digestive helminths. The plants were harvested in Benin, and their EO were obtained by hydrodistillation. The EO yield of extraction was 0.65% (w/w) of for Z. zanthoxyloides seeds and 0.05% (w/w) for N. laevis. The chemical compositions of the two EOs were analyzed by gas chromatography coupled with mass spectrometry. The major constituents of the EO from Z. zanthoxyloides consisted of the following compounds: γ-terpinene (18 %), undecane (15 %), valencene (8.3 %), decanal (8.3 %), and 3-carene (6.7 %). In contrast, the major constituents of the EO from N. laevis leaves consisted of the following compounds: β-caryophyllene (36 %) and eugenol (5.8 %). An egg-hatching inhibition (EHI) assay was developed and a larval migration inhibition assay was used on S. ratti to examine the effects of the EOs and to evidence their inhibitory concentrations (IC₅₀ and IC₉₀) values on this nematode. Furthermore, the toxicity of the two EOs on Vero cell line was evaluated. When tested on S. ratti egg hatching, the two EOs resulted in similar IC₅₀ values (19.5 and 18.2 μg/ml for Z. zanthoxyloides and N. laevis, respectively), which were about sevenfold higher than that of the control (thiabendazole, IC₅₀ = 2.5 μg/ml). Larval migration was inhibited at similar concentrations for: Z. zanthoxyloides (IC₅₀ = 46 μg/ml), N. laevis (IC₅₀ = 51 μg/ml), and the control [levamisole (IC₅₀ = 36 μg/ml)]. No cytotoxicity was found on Vero cells because both EOs had IC₅₀ values higher than 50 μg/ml. Therefore, we have concluded that the EOs from two plants, used in folk medicine, may contain compounds with anthelmintic activity and could be used as improved traditional medicines or, at least, as food additives in a combined treatment for the control of helminth infections.     

Effects of Panmede and various horse serum concentrations on the axenic cultivation ofEntamoeba histolytica strains in TPS-1 medium

We analyzed the influence of Panmede and horse serum concentrations on the growth of fiveEntamoeba histolytica strains (HK9, HM1, HM2, HM3 and HM38) axenically cultivated in TPS-1 medium. Panmede was evaluated by comparing the growth of strain HM1 in medium prepared with each of 15 Panmede lots; the yields ofE. histolytica trophozoites depended on the lot quality of Panmede, and their maximal values ranged from 8×10³ to 8.9×10⁴ amoebae/ml. The growth-promoting effect of eight lots of horse serum on strains HK9 and HM1 were studied using a single Panmede lot of good quality. Yields obtained with strain HK9 ranged from 8×10⁴ to 1.8×10⁵ amoebae/ml, whereas yields obtained with HM1 ranged from 3×10⁴ to 1.2×10⁵ amoebae/ml. Thus, the optimal serum concentration in TPS-1 medium that caused maximal growth ofE. histolytica cultures depended on the quality of the serum lot and proved to be specific for each of the fiveE. histolytica strains investigated. It ranged from 18% (v/v) for strain HM2 to 28% (v/v) for strain HM1. Our results reveal that the growth ofE. histolytica trophozoites in TPS-1 medium can be distinctly improved by selecting appropriate lots of Panmede and horse serum and using optimal serum concentrations.     

Anti-leishmania activity of semi-purified fraction of <Emphasis Type="Italic">Jacaranda puberula</Emphasis> leaves

The crude methanolic extract from leaves of Jacaranda puberula showed activity against Leishmania (Leishmania) amazonensis. The extract presented active against promastigote forms with an inhibitory concentration 50% (IC₅₀) value of 88.0 μg/ml, but only moderated activity against amastigote forms; however in higher concentrations the extract showed cytotoxic effects. The bio-guided chromatographic fractionation the crude methanolic extract against amastigotes yielded a fraction with an IC₅₀ value of 14.0 μg/ml (without cytotoxic activity) in relation to the crude extract (IC₅₀ value, 359.0 μg/ml). These data indicate that J. puberula leaves contain active compounds, which should be further investigated for the development of new potential drugs against cutaneous leishmaniasis.     

In vitro antiplasmodial activity and cytotoxicity of crude extracts and compounds from the stem bark of <Emphasis Type="Italic">Kigelia africana</Emphasis> (Lam.) Benth (Bignoniaceae)

In order to assess the potential of the stem bark of Kigelia africana (Lam.) Benth as source of new anti-malarial leads, n-hexane and ethyl acetate (EtOAc) extracts and four compounds isolated from the stem bark were screened in vitro against the chloroquine-resistant W-2 and two field isolates of Plasmodium falciparum using lactate dehydrogenase assay. The products were also tested for their cytotoxicity on LLC/MK2 monkey kidney cells. The EtOAc extract exhibited a significant antiplasmodial activity (IC₅₀ = 11.15 μg/mL on W-2; 3.91 and 4.74 μg/mL on field CAM10 and SHF4 isolates, respectively), whereas the n-hexane fraction showed a weak activity (IC₅₀ = 73.78 μg/mL on W-2 and 21.85 μg/mL on SHF4). Three out of the four compounds showed good activity against all the three different parasite strains (IC₅₀ < 5 μM). Specicoside exhibited the highest activity on W-2 (IC₅₀ = 1.54 μM) followed by 2β, 3β, 19α-trihydroxy-urs-12-en-28-oic acid (IC₅₀ = 1.60 μM) and atranorin (IC₅₀ = 4.41 μM), while p-hydroxycinnamic acid was the least active (IC₅₀ = 53.84 μM). The EtOAc extract and its isolated compounds (specicoside and p-hydroxycinnamic acid) were non-cytotoxic (CC₅₀ > 30 μg/mL), whereas the n-hexane extract and two of its products, atranorin and 2β, 3β, 19α-trihydroxy-urs-12-en-28-oic acid showed cytotoxicity at high concentrations, with the last one being the most toxic (CC₅₀ = 9.37 μg/mL). These findings justify the use of K. africana stem bark as antimalaria by traditional healers of Western Cameroon, and could constitute a good basis for further studies towards development of new leads or natural drugs for malaria.     

Antiplasmodial activity of botanical extracts against Plasmodium falciparum

The absence of a vaccine and the rampant resistance to almost all antimalarial drugs have accentuated the urgent need for new antimalarial drugs and drug targets for both prophylaxis and chemotherapy. The aim of the study was to discover effective plant extracts against Plasmodium falciparum. In the present study, the hexane, chloroform, ethyl acetate, acetone, and methanol extracts of Citrus sinensis (peel), Leucas aspera, Ocimum sanctum, Phyllanthus acidus (leaf), Terminalia chebula (seed) were tested for their antimalarial activity against chloroquine (CQ)-sensitive (3D7) strain of P. falciparum which was cultured following the candle-jar method. Antimalarial evaluations of daily replacement of culture medium containing CQ and different plant crude extracts were performed on 96-well plates at 37°C for 24 and 48 h. Parasitemia was determined microscopically on thin-film Giemsa-stained preparations. Plant extracts were tested for their cytotoxicity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on human laryngeal cancer cell line (HEp-2) and normal cell line (Vero). Out of the 25 extracts tested, six showed good (IC₅₀ 4.76–22.76 μg/mL), 15 exhibited moderate (IC₅₀ 31.42–88.03 μg/mL), while four displayed mild (IC₅₀ > 100 μg/mL) antiplasmodial activity. The leaf ethyl acetate and methanol extracts of L. aspera; ethyl acetate, acetone, and methanol extracts of P. acidus; and seed acetone extract of T. chebula had good antiplasmodial activity (IC₅₀ = 7.81, 22.76, 9.37, 14.65, 12.68, and 4.76 μg/mL) with selectivity indices 5.43, 2.04, 4.88, 3.35, 3.42, and 9.97 for HEp-2 and >5.79, >2.20, >11.75, >3.41, >3.94, and >7.38 for Vero cells, respectively. These analyses have revealed for the first time that the components present in the solvent extracts of L. aspera, P. acidus, and T. chebula have antiplasmodial activity. The high antiplasmodial activity observed make these plants good candidates for isolation of anti-protozoal compounds which could serve as new lead structures for drug development.     

In vitro and in vivo trypanocidal activity of native plants from the Yucatan Peninsula

Ethanol extracts of Senna villosa, Serjania yucatanensis, Byrsonima bucidaefolia, and Bourreria pulchra were evaluated for their in vitro activity against epimastigotes and trypomastigotes of Trypanosoma cruzi. Results showed that the leaf extracts of S. yucatanensis and B. pulchra were the most active against epimastigotes (IC₁₀₀ = 100 μg/mL) and trypomastigotes of T. cruzi (95% or more reduction in the number of parasites at 100 and 50 μg/mL). However, only the leaf extract of S. yucatanensis showed significant trypanocidal activity when tested in vivo, reducing 75% of the parasitemia in infected mice at 100 mg/kg. This same extract inhibited the egress of trypomastigotes from infected cells and proved not to be cytotoxic (IC₅₀ = 318.8 ± 2.3 μg/mL).     

In vitro evaluation of the effectiveness and cytotoxicity of meglumine antimoniate microspheres produced by spray drying against Leishmania infantum

The aim of the present study was to evaluate the in vitro activity and cytotoxicity of meglumine antimoniate microspheres produced by spray drying on Leishmania infantum and the effect of the excipients used in them. The parasite strain shows sensitivity to the meglumine antimoniate microspheres prepared. All the antimony IC₅₀ values from encapsulated meglumine antimoniate (3.80 ± 0.34 to 9.53 ± 0.70 μg SbV/ml for promastigotes assay) are considerably lower compared to the mean value of IC₅₀ in Glucantime solution (112 ± 12.74 μg SbV/ml). Interesting IC₅₀ values for the excipient chitosan (112.64 ± 0.53 mg/ml for promastigotes and 100.81 ± 26.45 mg/ml for amastigotes) were obtained (without cytotoxic activity), whereas the rest of the excipients did not show any activity. This new delivery system could offer a new pharmacological tool for the treatment of leishmaniosis that reduces the doses required, lowering toxic side effects because of meglumine antimoniate.     

In vitro antimalarial activity of medicinal plant extracts against Plasmodium falciparum

Malaria is a major global public health problem, and the alarming spread of drug resistance and limited number of effective drugs now available underline how important it is to discover new antimalarial compounds. In the present study, ten plants were extracted with ethyl acetate and methanol and tested for their antimalarial activity against chloroquine (CQ)-sensitive (3D7) and CQ-resistant (Dd2 and INDO) strains of Plasmodium falciparum in culture using the fluorescence-based SYBR Green assay. Plant extracts showed moderate to good antiparasitic effects. Promising antiplasmodial activity was found in the extracts from two plants, Phyllanthus emblica leaf 50% inhibitory concentration (IC₅₀) 3D7: 7.25 μg/mL (ethyl acetate extract), 3.125 μg/mL (methanol extract), and Syzygium aromaticum flower bud, IC₅₀ 3D7:13 μg/mL, (ethyl acetate extract) and 6.25 μg/mL (methanol extract). Moderate activity (30–75 μg/mL) was found in the ethyl acetate and methanol extracts of Abrus precatorius (seed) and Gloriosa superba (leaf); leaf ethyl acetate extracts of Annona squamosa and flower of Musa paradisiaca. The above mentioned plant extracts were also found to be active against CQ-resistant strains (Dd2 and INDO). Cytotoxicity study with P. emblica leaf and S. aromaticum flower bud, extracts showed good therapeutic indices. These results demonstrate that leaf ethyl acetate and methanol extracts of P. emblica and flower bud extract of S. aromaticum may serve as antimalarial agents even in their crude form. The isolation of compounds from P. emblica and S. aromaticum seems to be of special interest for further antimalarial studies.     

Cytopathogenicity ofEntamoeba histolytica: Trophozoite homogenates modulate DNA synthesis in a mammalian cell line

We examined the effect of total trophozoite homogenates from four axenized strains ofEntamoeba histolytica (HK9, HM1, HM2, and HM3) on the DNA synthesis of subconfluent cultures of Chinese hamster ovary (CHO) cells incubated at low (0.1%) serum concentration. HM1, HM2, and HM3 extracts increased [³H]thymidine incorporation to acidinsoluble material in CHO cells up to a maximum of 2.5, 1.5, and 1.5 times respectively, at doses of amebal protein ranging from 16 to 125 g/ml. HM1 and HM2 extracts at doses higher than those causing maximal stimulation, and HM3 and HK9 extracts above 250 g protein per ml, progressively inhibited [³H]thymidine incorporation by CHO cells at a strain-specific rate. The extracts with both the most potent stimulatory and inhibitory effects were those from HM1 and HM2, also the most virulent strains. This strain-specific ability of amebal products to modulate cell DNA synthesis may play a significant role in amebal virulence.     

Antileishmanial potential of a marine sponge, <Emphasis Type="Italic">Haliclona exigua</Emphasis> (Kirkpatrick) against experimental visceral leishmaniasis

In this study, we are reporting antileishmanial activity of a marine sponge Haliclona exigua, belonging to phylum Porifera. The crude methanol extract and its three fractions were tested both in vitro and in vivo. The crude extract exerted almost complete inhibition of promastigotes at 50 μg/ml and 76.4 ± 6.5% inhibition of intracellular amastigotes at 100 μg/ml concentration with IC₅₀ values of 18.6 μg/ml and 47.2 μg/ml, respectively. When administered to Leishmania donovani infected hamsters at a dose of 500 mg/kg × 5, p.o., it resulted in 72.2 ± 10.4% inhibition of intracellular amastigotes. At a lower dose (250 mg/kg), it exhibited 43.9 ± 5.1% inhibition. Among the fractions, highest antileishmanial activity both in vitro (>90%) and in vivo (60.9 ± 18.3%) was observed in n-butanol (soluble) fraction with IC₅₀ values of 8.2 μg/ml and 31.2 μg/ml against promastigotes and intracellular amastigotes, respectively. Hexane fraction also showed comparatively good activity against both the stages of parasites in vitro but was moderately active in leishmania-infected hamsters. Chloroform fraction resulted in 45 ± 10.2% inhibition in vivo at a dose of 500 mg/kg × 5, p.o., whereas it was inactive in vitro. n-Butanol (insoluble) fraction was inactive both in vitro and in vivo. Araguspongin C, an alkaloid isolated from n-butanol (soluble) fraction exhibited moderate inhibition of promastigotes and intracellular amastigotes at 100 μg/ml but showed weak antileishmanial action in vivo. Our findings indicate that this marine sponge has the potential to provide new lead toward development of an effective antileishmanial agent and, hence, calls for more exhaustive studies for exploiting the vast world of marine resources to combat the scourge of several parasitic diseases.     

In vitro activities of plant extracts on human Loa loa isolates and cytotoxicity for eukaryotic cells

Loa loa, a filarial worm, can cause fatal encephalitis in humans. In an attempt to find alternatives to the standard treatments (ivermectin and diethylcarbamazine citrate), we tested 12 methanolic extracts of nine traditional plant remedies. The extracts (100–0.09 μg/ml) were incubated with 20 Loa loa microfilariae isolated from patients at 37°C with 5% CO₂ in modified Eagle’s medium supplemented with 10% fetal serum and antibiotics. Activity was evaluated 120 h later by counting live microfilariae under a microscope. Cytotoxicity for eukaryotic cells was estimated by measuring 3-[4,5-dimethylthiazol-2-yl]-2-5 diphenyl tetrazolium bromide transformation to formazan at 450 nM in a spectrophotometer. The plants tested were Lophira alata, Greenwayodendron suaveolens, Uapaca togoensis, Zanthoxylum heitzii, Peperomia pellucida, Piptadeniastrum africanum, Petersianthus macrocarpus, Vernonia conferta, and Vernonia hymenolepis. Chemical screening showed that most of the extracts contained reducing sugars, tannin or polyphenols, sterols or triterpenes, saponosides, and alkaloids. None contained carotinoids and few contained flavonoids. The 50% lethal concentration ranged from 0.22 to 70.28 μg/ml, while the 50% inhibitory concentration for eukaryotic cells (IC₅₀) ranged from 8.52 to 119.52 μg/ml. Extracts of P. macrocarpus (selectivity index = 72.16), P. africanum (13.69), Z. heitzii (12.11), and L. alata (9.26) were highly selective for L. loa.     

Purification and immunologic characterization of a 30-kDa cysteine proteinase ofEntamoeba histolytica

A 30-kDa cysteine proteinase was purified from extracts of axenically grown trophozoites ofEntamoeba histolytica strain HM1:IMSS. The purification procedure involved two consecutive chromatographic steps. Sequence analysis revealed high similarity with histolysin and with other 27-kDa cysteine proteinase. Western-blot analysis using F(ab)₂ fragments of a polyclonal antibody raised against the purified enzyme revealed that when the amebic extract was prepared in the absence of proteinase inhibitors there were many positive bands ranging in relative molecular weight from 115 to 12.5kDa, but when the extract was prepared in the presence of proteinase inhibitors there was only a single 30-kDa positive band. Similar results were obtained with immunoprecipitates. This phenomenon would suggest the formation of multimer aggregates of the 30-kDa cysteine proteinase after partial proteolysis.