Giardia lamblia: the effects of extracts and fractions from Mentha x piperita Lin. (Lamiaceae) on trophozoites

Giardia lamblia is a parasite that causes giardiasis in humans and other mammals. The common treatment includes different classes of drugs, which were described to produce unpleasant side effects. Mentha x piperita, popularly known as peppermint, is a plant that is frequently used in the popular medicine to treat gastrointestinal symptoms. We examined the effects of crude extracts and fractions from peppermint against G. lamblia (ATCC 30888) on the basis of trophozoite growth, morphology and adherence studies. The methanolic, dichloromethane and hexanic extracts presented IC(50) values of 0.8, 2.5 and 9.0microg/ml after 48h of incubation, respectively. The aqueous extract showed no effect against the trophozoites with an IC(50)>100microg/ml. The aqueous fraction presented a moderate activity with an IC(50) of 45.5microg/ml. The dichloromethane fraction showed the best antigiardial activity, with an IC(50) of 0.75microg/ml after 48h of incubation. The morphological and adhesion assays showed that this fraction caused several alterations on plasma membrane surface of the parasite and inhibited the adhesion of G. lamblia trophozoites. Cytotoxic assays showed that Mentha x piperita presented no toxic effects on the intestinal cell line IEC-6. Our results demonstrated antigiardial activity of Mentha x piperita, indicating its potential value as therapeutic agent against G. lamblia infections.     

In vitro and in vivo leishmanicidal activity of Dysoxylum binectariferum and its fractions against Leishmania donovani

The leishmanicidal effect of crude ethanolic extract of stem bark of Dysoxylum binectariferum and its fractions has been investigated against Leishmania donovani, the causative agent of visceral leishmaniasis. Ethanolic extract was lethal to promastigotes as well as amastigote forms in macrophage system at the concentration of 100 microg/ml. Chloroform fraction significantly inhibited promastigote multiplication and was also active against amastigotes in infected J774A.1 macrophages at 100 microg/ml. Hexane fraction was moderately active and the other fractions were inactive against both the forms. When tested in vivo in hamsters, ethanolic extract was toxic at 500 mg/kg whereas exhibited marginal activity (67.7+/-5.3%) at 250 mg/kg x 5, p.o. on day 7 post treatment (p.t.) which increases slightly (69+/-4.7) by day 30 p.t. Chloroform and n-hexane fractions exhibited 64.3+/-4% and 47.8+/-4.6% parasite inhibition at the dose of 100 mg/kg x 5 p.o., respectively. The pure compound, rohitukine, obtained from chloroform fraction showed weaker in vitro activity and was ineffective in infected hamsters. The lead potential of this plant need further investigations.     

The effect of blueberry extracts on Giardia duodenalis viability and spontaneous excystation of Cryptosporidium parvum oocysts, in vitro

The protozoan parasites Giardia duodenalis and Cryptosporidium parvum are common causes of diarrhoea, worldwide. Effective drug treatment is available for G. duodenalis, but with anecdotal evidence of resistance or reduced compliance. There is no effective specific chemotherapeutic intervention for Cryptosporidium. Recently, there has been renewed interest in the antimicrobial properties of berries and their phenolic compounds but little work has been done on their antiparasitic actions. The effect of various preparations of blueberry (Vaccinium myrtillus) extract on G. duodenalis trophozoites and C. parvum oocysts were investigated. Pressed blueberry extract, a polyphenolic-rich blueberry extract, and a commercially produced blueberry drink (Bouvrage) all demonstrated antigiardial activity. The polyphenol-rich blueberry extract reduced trophozoite viability in a dose dependent manner. At 167 μg ml⁻¹, this extract performed as well as all dilutions of pressed blueberry extract and the Bouvrage beverage (9.6 ± 2.8% live trophozoites remaining after 24 h incubation). The lowest dilution of blueberry extract tested (12.5% v/v) contained >167 μg ml⁻¹ of polyphenolic compounds suggesting that polyphenols are responsible for the reduced survival of G. duodenalis trophozoites. The pressed blueberry extract, Bouvrage beverage and the polyphenolic-rich blueberry extract increased the spontaneous excystation of C. parvum oocysts at 37 °C, compared to controls, but only at a dilution of 50% Bouvrage beverage, equivalent to 213 μg ml⁻¹ gallic acid equivalents in the polyphenolic-rich blueberry extract. Above this level, spontaneous excystation is decreased. We conclude that water soluble extracts of blueberries can kill G. duodenalis trophozoites and modify the morphology of G. duodenalis and C. parvum.     

Anti-Giardia activity of Syzygium aromaticum essential oil and eugenol: effects on growth, viability, adherence and ultrastructure

The present work evaluates the anti-Giardia activity of Syzygium aromaticum and its major compound eugenol. The effects were evaluated on parasite growth, adherence, viability and ultrastructure. S. aromaticum essential oil (IC₅₀ = 134 μg/ml) and eugenol (IC₅₀ = 101 μg/ml) inhibited the growth of G. lamblia. The essential oil inhibited trophozoites adherence since the first hour of incubation and was able to kill almost 50% of the parasites population in a time dependent manner. The eugenol inhibited G. lamblia trophozoites adherence since the third hour and not induce cell lyses. The main morphological alterations were modifications on the cell shape, presence of precipitates in the cytoplasm, autophagic vesicles, internalization of flagella and ventral disc, membrane blebs, and intracellular and nuclear clearing. Taken together, our findings lead us to propose that eugenol was responsible for the anti-giardial activity of the S. aromaticum essential oil and both have potential for use as therapeutic agents against giardiasis.     

Effects of Brazilian propolis on Leishmania amazonensis

Leishmaniasis, an endemic parasitosis that leads to chronic cutaneous, mucocutaneous or visceral lesions, is part of those diseases, which still requires improved control tools. Propolis has shown activities against different bacteria, fungi, and parasites. In this study we investigated the effect of four ethanolic extracts of typified propolis collected in different Brazilian states, on Leishmania amazonensis performing assays with promastigote forms, extracellular amastigotes, and on infected peritoneal macrophages. Ethanolic extracts of all propolis samples (BRG, BRPG, BRP-1, and BRV) were capable to reduce parasite load as monitored by the percentage of infected macrophages and the number of intracellular parasites. BRV sample called red propolis, collected in the state of Alagoas, and containing high concentration of prenylated and benzophenones compounds, was the most active extract against L. amazonensis. The anti-Leishmania effect of BRV sample was increased in a concentration and time dependent manner. BRV treatment proved to be non-toxic to macrophage cultures. Since BRV extract at the concentration of 25 microg/ml reduced the parasite load of macrophages while presented no direct toxic to promastigotes and extracellular amastigotes, it was suggested that constituents of propolis intensify the mechanism of macrophage activation leading to killing of L. amazonensis. Our results demonstrate, for the first time, that ethanolic extracts of Brazilian propolis reduce L. amazonensis infection in macrophages, and encourage further studies of this natural compound in animal models of leishmaniasis.     

Anticariogenic activity of macelignan isolated from Myristica fragrans (nutmeg) against Streptococcus mutans

The occurrence of dental caries is mainly associated with oral pathogens, especially cariogenic Streptococcus mutans. Preliminary antibacterial screening revealed that the extract of Myristica fragrans, widely cultivated for the spice and flavor of foods, possessed strong inhibitory activity against S. mutans. The anticariogenic compound was successfully isolated from the methanol extract of M. fragrans by repeated silica gel chromatography, and its structure was identified as macelignan by instrumental analysis using 1D-NMR, 2D-NMR and EI-MS. The minimum inhibitory concentration (MIC) of macelignan against S. mutans was 3.9 microg/ml, which was much lower than those of other natural anticariogenic agents such as 15.6 microg/ml of sanguinarine, 250 microg/ml of eucalyptol, 500 microg/ml of menthol and thymol, and 1000 microg/ml of methyl salicylate. Macelignan also possessed preferential activity against other oral microorganisms such as Streptococcus sobrinus, Streptococcus salivarius, Streptococcus sanguis, Lactobacillus acidophilus and Lactobacillus casei in the MIC range of 2-31.3 microg/ml. In particular, the bactericidal test showed that macelignan, at a concentration of 20 microg/ml, completely inactivated S. mutans in 1 min. The specific activity and fast-effectiveness of macelignan against oral bacteria strongly suggest that it could be employed as a natural antibacterial agent in functional foods or oral care products.     

Isolation of pure compound R/J/3 from Pluchea indica (L.) Less. and its anti-amoebic activities against Entamoeba histolytica

The plant Pluchea indica is known for its anti-inflammatory, anti-ulcer, anti-pyretic, hypoglycemic, diuretic and anti-microbial activities besides many other pharmacological activities. We have isolated and purified seven compounds from the methanolic root extract of this plant by column chromatography. The compounds were identified by spectroscopic analyses. The anti-amoebic activities of the pure compound R/J/3 was investigated against the HM1 strain of Entamoeba histolytica. The compound, R/J/3 showed the most pronounced anti-proliferative activity at a dose of 50 microg/ml. It also showed a marked activity on cell lysis of trophozoites, 4h after administration. The cell lytic activity was compared with metronidazole (5 microg/ml) as positive control.     

Chemical compositions and antimicrobial activities of four different Anatolian propolis samples

Propolis means a gum that is gathered by bees from various plants. It is known for its biological properties, having antibacterial, antifungal and healing properties. The aims of this study were to evaluate the antimicrobial activity of four different Anatolian propolis samples on different groups of microorganisms including some oral pathogens and comparison between their chemical compositions. Ethanol extracts of propolis (EEP) were prepared from four different Anatolian propolis samples and examined whether EEP inhibit the growth of the test microorganisms or not. For the antimicrobial activity assays, minimum inhibitory concentrations (MIC) were determined by using macrodilution method. The MIC values of the most effective propolis (TB) were 2 microg/ml for Streptococcus sobrinus and Enterococcus faecalis, 4 microg/ml for Micrococcus luteus, Candida albicans and C. krusei, 8 microg/ml for Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis and Enterobacter aerogenes, 16 microg/ml for Escherichia coli and C. tropicalis and 32 microg/ml for Salmonella typhimurium and Pseudomonas aeruginosa. The chemical compositions of EEP's were determined by high-temperature high-resolution gas chromatography coupled to mass spectrometry. The main compounds of four Anatolian propolis samples were flavonoids such as pinocembrin, pinostropin, isalpinin, pinobanksin, quercetin, naringenin, galangine and chrysin. Although propolis samples were collected from different regions of Anatolia all showed significant antimicrobial activity against the Gram positive bacteria and yeasts. Propolis can prevent dental caries since it demonstrated significant antimicrobial activity against the microorganisms such as Streptococcus mutans, Streptococcus sobrinus and C. albicans, which involves in oral diseases.     

Scuticociliate cysteine proteinases modulate turbot leucocyte functions

The effects exerted by cysteine proteinases isolated from the histiophagous ciliate Philasterides dicentrarchi on the phagocytic functions of turbot pronephric leucocytes (PL) were investigated. The enzymes were tested at concentrations of 125, 250 and 500 microg ml(-1), and it was found that the viability of the leucocytes was not affected after treatment for 24h. Leucocyte migration was inhibited by the cysteine proteinases in a dose-dependent manner, whereas the ascitic fluid obtained from turbot experimentally infected with P. dicentrarchi induced high chemotactic activity in the turbot PL. The proteinases did not affect yeast cell phagocytosis but increased intracellular production of the superoxide anion (O2(-)). Stimulation with the proteinases did not alter the PGE2 levels in supernatants from 24-h cultures of PL, however, beta-glucans (100 microg ml(-1)) provoked a large increase in PGE2 levels, which were inhibited after addition of 10 microg ml(-1) of indomethacin, a non-selective inhibitor of COX2 enzymatic activity. The mean PGE2 level in ascitic fluid from turbot, experimentally infected with P. dicentrarchi, was 500 pg ml(-1), and the addition of low levels of PGE2 (62.5 pg ml(-1)) to PL cultures stimulated O2(-) production, although addition of PGE2 at concentrations higher than 250 pg ml(-1) blocked the increase in stimulation. Addition of cysteine proteinases to 24-h cultures of PL also increased mRNA levels in the pro-inflammatory cytokine interleukin-1beta. The results revealed the capacity of cysteine proteinases isolated from P. dicentrarchi to modulate the innate immune response of turbot, which together with the inflammation mediators produced during infection, may play an important role in pathogenesis of the disease and in the survival of the parasite.     

Antioxidant, cyclooxygenase and topoisomerase inhibitory compounds from Apium graveolens Linn. seeds

Cyclooxygenase inhibitory and antioxidant bioassay-directed extraction and purification of celery seeds yielded sedanolide (1), senkyunolide-N (2), senkyunolide-J (3), 3-hydroxymethyl-6-methoxy-2,3-dihydro-1H-indol-2-ol (4), L-tryptophan (6), and 7-[3-(3,4-dihydroxy-4-hydroxymethyl-tetrahydro-furan-2-yloxy)-4,5-dihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy]-5-hydroxy-2-(4-hydroxy-3-methoxy-phenyl)-chromen-4-one (7). The structures of compounds 1-7 were determined using spectroscopic methods. Compound 4 is reported here for the first time. At 250 pg ml(-1), compounds 1-4, 6 and 7 displayed prostaglandin H endoperoxide synthase-I (COX-I) and prostaglandin H endoperoxide synthase-II (COX-II) inhibitory activities at pH 7. The acetylated product (5) of compound 4 also inhibited COX-I and COX-II enzymes when tested at 250 microg ml(-1). Compounds 6 and 7 exhibited good antioxidant activity at concentrations of 125 and 250 microg ml(-1). Only compounds 1-3 exhibited topoisomerase-I and -II enzyme inhibitory activity at concentrations of 100, 200 and 200 microg ml(-1), respectively.     

Antimutagenicity of a suberin extract from Quercus suber cork

The possible protective effect of a suberin extract from Quercus suber cork on acridine orange (AO)-, ofloxacin- and UV radiation-induced mutagenicity (bleaching activity) in Euglena gracilis was examined. To our knowledge, the present results are the first attempt to analyse suberin in relation to mutagenicity of some chemicals. Suberin exhibits a significant dose-dependent protective effect against AO-induced mutagenicity and the concentration of 500 μg/ml completely eliminates the Euglena-bleaching activity of AO. The mutagenicity of ofloxacin is also significantly reduced in the presence of suberin (125, 250 and 500 μg/ml). However, the moderate protective effect of suberin on UV radiation-induced mutagenicity was observed only at concentrations 500 and 1000 μg/ml. Our data shows that suberin extract from Q. suber cork possess antimutagenic properties and can be included in the group of natural antimutagens acting in a desmutagenic manner.     

In vitro antimicrobial activity of propolis samples from different geographical origins against certain oral pathogens

Propolis is an agent having antimicrobial properties, however, its composition can vary depending on the area where it is collected. In the present study, the antimicrobial activity of five propolis samples, collected from four different regions in Turkey and from Brazil, against nine anaerobic strains was evaluated. Ethanol extracts of propolis (EEP) were prepared from propolis samples and we determined minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of EEP on the growth of test microorganisms by using agar dilution method. All strains were susceptible and MIC values ranged from 4 to 512 microg/ml for propolis activity. Propolis from Kazan-Ankara showed most effective MIC values to the studied microorganisms. MBC values of Kazan-Ankara EEP samples were ranged from 8 to 512 microg/ml. Death was observed within 4 h of incubation for Peptostreptococcus anaerobius and micros and Lactobacillus acidophilus and Actinomyces naeslundii, while 8 h for Prevotella oralis and Prevotella melaninogenica and Porphyromonas gingivalis, 12 h for Fusobacterium nucleatum, 16 h for Veillonella parvula. It was shown that propolis samples were more effective against Gram positive anaerobic bacteria than Gram negative ones. The organic chemical compositions of EEPs were determined by high-resolution gas chromatography coupled to mass spectrometry (GC-MS). The main compounds of EEPs were flavonoids such as pinobanksin, quercetin, naringenin, galangine, chrysin and aromatic acids such as cafeic acid. Because of increased antimicrobial resistance, propolis may be kept in mind in the treatment of oral cavity diseases.     

Periodic appearance of a predominant variant antigen type during a chronic Giardia lamblia infection in a mouse model

Giardia lamblia (syn. Giardia duodenalis, Giardia intestinalis) infections are associated with continuous antigenic variation of the parasite which is mediated by the parasite's major surface antigen, named variant surface protein. Offspring mice and corresponding mothers were infected with G. lamblia clone GS/M-83-H7 (expressing variant surface protein H7) and various parameters of this infection were assessed in a long-term follow-up investigation. Our experimentation revealed that variant surface protein H7-type trophozoites were replaced by new variant-type trophozoites during the early stage of infection (around day 8 p.i.), but the original variant-type re-emerged at at least two time-points during the later stages of infection (at days 22 and 42 p.i.). Such periods of variant surface protein H7-type trophozoite re-expansion were accompanied by transient production of intestinal IgA against variant-specific epitopes on a 314-aa N-terminal region of variant surface protein H7. At late stages of infection (between days 42 and 200 p.i.), most mice produced intestinal IgA against both variant surface protein H7 and other antigens of the parasite. At these stages, infection seemed to be resolved in most mice, but occasional reappearance of relatively high (at day 64 p.i.) or at least detectable (at days 80 and 120 p.i.) amounts of intestinal parasites indicated that G. lamblia GS/M-83-H7 infections in mice may enter into a latent chronic phase which is interrupted by sporadic breakthroughs of parasite growth.     

Giardia lamblia: ultrastructural study of the in vitro effect of benzimidazoles.

Axenically grown Giardia lamblia trophozoites treated with low concentrations of the benzimidazole carbamates albendazole and mebendazole detach from glass culture tubes and lose viability. Scanning electron microscopic observations revealed that these drugs produce grotesque modifications of the cell shape of the parasite and disassembly of the adhesive disc. Transmission electron microscopy showed several stages of the fragmentation of adhesive discs with dispersion of microtubules and microribbons in the cytoplasm. Flagella appeared undamaged. In drug-treated trophozoites electron-dense precipitates were selectively deposited on microtubules and microribbons. The results indicate that the antigiardial effect of benzimidazoles is the result of binding to microtubules and subsequent alterations of the cytoskeleton. The electron microscopic observations also suggest that the drugs may bind to microribbon components of the adhesive disc, possibly giardin proteins.     

The effect of blueberry extracts on Giardia duodenalis viability and spontaneous excystation of Cryptosporidium parvum oocysts, in vitro

The protozoan parasites Giardia duodenalis and Cryptosporidium parvum are common causes of diarrhoea, worldwide. Effective drug treatment is available for G. duodenalis, but with anecdotal evidence of resistance or reduced compliance. There is no effective specific chemotherapeutic intervention for Cryptosporidium. Recently, there has been renewed interest in the antimicrobial properties of berries and their phenolic compounds but little work has been done on their antiparasitic actions. The effect of various preparations of blueberry (Vaccinium myrtillus) extract on G. duodenalis trophozoites and C. parvum oocysts were investigated. Pressed blueberry extract, a polyphenolic-rich blueberry extract, and a commercially produced blueberry drink (Bouvrage) all demonstrated antigiardial activity. The polyphenol-rich blueberry extract reduced trophozoite viability in a dose dependent manner. At 167 microgml(-1), this extract performed as well as all dilutions of pressed blueberry extract and the Bouvrage beverage (9.6+/-2.8% live trophozoites remaining after 24h incubation). The lowest dilution of blueberry extract tested (12.5% v/v) contained >167 microgml(-1) of polyphenolic compounds suggesting that polyphenols are responsible for the reduced survival of G. duodenalis trophozoites. The pressed blueberry extract, Bouvrage beverage and the polyphenolic-rich blueberry extract increased the spontaneous excystation of C. parvum oocysts at 37 degrees C, compared to controls, but only at a dilution of 50% Bouvrage beverage, equivalent to 213 microgml(-1) gallic acid equivalents in the polyphenolic-rich blueberry extract. Above this level, spontaneous excystation is decreased. We conclude that water soluble extracts of blueberries can kill G. duodenalis trophozoites and modify the morphology of G. duodenalis and C. parvum.     

Giardia lamblia: Ultrastructural Study of the In Vitro Effect of Benzimidazoles

ABSTRACT Axenically grown Giardia lamblia trophozoites treated with low concentrations of the benzimidazole carbamates albendazole and mebendazole detach from glass culture tubes and lose viability. Scanning electron microscopic observations revealed that these drugs produce grotesque modifications of the cell shape of the parasite and disassembly of the adhesive disc. Transmission electron microscopy showed several stages of the fragmentation of adhesive discs with dispersion of microtubules and microribbons in the cytoplasm. Flagella appeared undamaged. In drug-treated trophozoites electron-dense precipitates were selectively deposited on microtubules and microribbons. The results indicate that the antigiardial effect of benzimidazoles is the result of binding to microtubules and subsequent alterations of the cytoskeleton. The electron microscopic observations also suggest that the drugs may bind to microribbon components of the adhesive disc, possibly giardin proteins.     

Giardia lamblia: evaluation of the in vitro effects of nocodazole and colchicine on trophozoites

Giardia lamblia is the most commonly detected parasite in the intestinal tract of humans and other mammals causing giardiasis. Giardia presents several cytoskeletal structures with microtubules as major components such as the ventral adhesive disk, eight flagella axonemes, the median body and funis. Many drugs have already been tested as antigiardial agents, such as albendazole and mebendazole, which act by specifically inhibiting tubulin polymerization and hence microtubule assembly. In the present work, we used the microtubule inhibitors nocodazole and colchicine in order to investigate their direct and indirect effects on Giardia ultrastructure and attachment to the glass surface, respectively. Axenically grown G. lamblia trophozoites were treated with nocodazole or colchicine for different time intervals and analyzed by light and electron microscopy. It was observed that trophozoites became completely misshapen, detached from the glass surface and failed to complete cell division. The main alterations observed included disc fragmentation, presence of large vacuoles, and appearance of electrondense deposits made of tubulin. The cytokinesis was blocked, but not the karyokinesis, and membrane blebs were observed. These findings show that Giardia behavior and cytoskeleton are clearly affected by the commonly used microtubule targetting agents colchicine and nozodazole.     

Effect of Mexican propolis extracts from Apis mellifera on Candida albicans in vitro growth

Propolis is a resinous substance collected by bees (Apis mellifera) from different trees and bushes. Due to its antifungal, antibacterial, antiviral and antiparasitic properties, it has continued to be very popular throughout the time showing variable activity depending on its geographical origin. In Mexico, information about this product is very limited. The aim of this work was to evaluate the antifungal activity of four propolis ethanolic extracts from three different Mexican states, and four commercial extracts on Candida albicans growth. A reference strain (ATCC 10231) and 36 clinical isolates of C. albicans were used. The Minimal Inhibitory Concentration (MIC) was determined by the dilution on agar method. Growth curves on Sabouraud Dextrose broth with and without different propolis ethanolic extracts concentrations were performed. In addition, whether the effect was fungistatic or fungicide was determined. The propolis ethanolic extract obtained from Cuautitlán Izcalli, State of Mexico, showed the best biological activity, inhibiting 94.4% from the clinical isolates at 0.8 mg/ml; the reference strain was inhibited at 0.6 mg/ml. The propolis effect was fungistatic in low concentrations and fungicide in concentrations higher to MIC. The Mexican propolis ethanolic extract could be further investigated for its alternative use for the treatment of some C. albicans infections.     

Preparation of Water and Ethanolic Extracts of Propolis and Evaluation of the Preparations

Propolis was extracted using water and various concentrations of ethanol as solvents. The extracts were investigated by measurement of absorption spectrum with a UV spectrophotometer, reversed phase-high pressure thin-layer chromatography and reversed phase-HPLC. Maximum absorption of all extracts was 290 nm, resembling flavonoid compounds, and the 80% ethanolic extract showed highest absorption at 290 nm. The most isosakuranetin, quercetin, and kaempferol were extracted from mixtures of propolis and 60% ethanol, while 70% ethanol extracted the most pinocembrin and sakuranetin, but 80% ethanol extracted more kaempferide, acacetin, and isorhamnetin from propolis. The 60 to 80% ethanolic extracts of propolis strongly inhibited microbial growth and 70 and 80% ethanolic extracts had the greatest antioxidant activity and 80% ethanolic extract strongly inhibited hyaluronidase activity.