In vitro antidermatophytic activity of Otacanthus azureus (Linden) Ronse essential oil alone and in combination with azoles.

AIMS: We determined the chemical composition and investigated the antifungal activity of Otacanthus azureus (Linden) Ronse essential oil (EO) against a range of dermatophytes alone or in combination with azole antifungals. METHODS AND RESULTS: Aerial parts of the plant were steam-distilled and the obtained oil was analysed by gas chromatography/mass spectrometry and (1) H-NMR. It was shown to be largely composed of sesquiterpenes, with the main component being ß-copaen-4-α-ol. Using broth microdilution techniques, this oil was found to have remarkable in vitro antifungal activities. Minimum inhibitory concentrations as low as 4 µg ml(-1) were recorded. The analysis of the combined effect of the O. azureus EO with azoles using chequerboard assays revealed a synergism between the EO and ketoconazole, fluconazole or itraconazole against Trichophyton mentagrophytes. Notably, the O. azureus essential oil showed low cytotoxicity to VERO cells. CONCLUSIONS: The O. azureus essential oil alone or in combination with azoles is a promising antifungal agent in the treatment for human dermatomycoses caused by filamentous fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: There is much interest in the study of essential oils for the discovery of new antimicrobial drugs. This study has highlighted the antidermatophytic activity of the O. azureus EO.

Erythrocyte stages of Plasmodium falciparum exhibit a high nitric oxide synthase (NOS) activity and release an NOS-inducing soluble factor.

Nitric oxide (NO), a highly diffusible cellular mediator involved in a wide range of biological effects, has been indicated as one of the cytotoxic agents released by leukocytes to counteract malaria infection. On the other hand, NO has been implicated as a mediator of the neuropathological symptoms of cerebral malaria. In such circumstances NO production has been thought to be induced in host tissues by host-derived cytokines. Here we provide evidence for the first time that human red blood cells infected by Plasmodium falciparum (IRBC) synthesize NO. The synthesis of NO (measured as citrulline and nitrate production) appeared to be very high in comparison with human endothelial cells; no citrulline and nitrate production was detectable in noninfected red blood cells. The NO synthase (NOS) activity was very high in the lysate of IRBC (while not measurable in that of normal red blood cells) and was inhibited in a dose-dependent way by three different NOS inhibitors (L-canavanine, NG-amino-L-arginine, and NG-nitro-L-arginine). NOS activity in P. falciparum IRBC is Ca++ independent, and the enzyme shows an apparent molecular mass < 100 kD, suggesting that the parasite expresses an isoform different from those found in mammalian cells. IRBC release a soluble factor able to induce NOS in human endothelial cells. Such NOS-inducing activity is not tissue specific, is time and dose dependent, requires de novo protein synthesis, and is probably associated with a thermolabile protein having a molecular mass > 100 kD. Our data suggest that an increased NO synthesis in P. falciparum malaria can be directly elicited by soluble factor(s) by the blood stages of the parasite, without necessarily requiring the intervention of host cytokines.

Antimalarial activity of simalikalactone E, a new quassinoid from Quassia amara L. (Simaroubaceae).

We report the isolation and identification of a new quassinoid named simalikalactone E (SkE), extracted from a widely used Amazonian antimalarial remedy made out of Quassia amara L. (Simaroubaceae) leaves. This new molecule inhibited the growth of Plasmodium falciparum cultured in vitro by 50%, in the concentration range from 24 to 68 nM, independently of the strain sensitivity to chloroquine. We also showed that this compound was able to decrease gametocytemia with a 50% inhibitory concentration sevenfold lower than that of primaquine. SkE was found to be less toxic than simalikalactone D (SkD), another antimalarial quassinoid from Q. amara, and its cytotoxicity on mammalian cells was dependent on the cell line, displaying a good selectivity index when tested on nontumorogenic cells. In vivo, SkE inhibited murine malaria growth of Plasmodium vinckei petteri by 50% at 1 and 0.5 mg/kg of body weight/day, by the oral or intraperitoneal routes, respectively. The contribution of quassinoids as a source of antimalarial molecules needs therefore to be reconsidered.

Ethnopharmacology and malaria: new hypothetical leads or old efficient antimalarials?

New treatments are urgently needed to curb and eradicate malaria in developing countries. As most people living in malarial endemic areas use traditional medicine to fight this disease, why have new treatments not emerged recently from ethnopharmacology-oriented research? The rationale and limitations of the ethnopharmacological approach are discussed in this paper, focusing on ethnopharmacology methodologies and techniques used for assessing botanical samples for their antimalarial properties. Discrepancies often observed between strong ethnopharmacological reputation and laboratory results are discussed, as well as new research perspectives.

Quassia amara L. (Simaroubaceae) leaf tea: effect of the growing stage and desiccation status on the antimalarial activity of a traditional preparation.

In French Guiana, Quassia amara L. (Simaroubaceae) leaf tea is a well-known widely used traditional antimalarial remedy. Impact of the vegetal sampling condition on in vivo and in vitro antimalarial activity was assessed. Traditional infusions were prepared with juvenile or mature leaves, both either fresh or dried. Results showed that growing stage and freshness of vegetal material exert a striking effect on antimalarial activity, both in vitro and in vivo. By far, leaf tea made from fresh juvenile (FJ) Quassia amara leaves was the most active. In vitro, active component (simalikalactone D) concentration correlates biological activities, although unexplained subtle variations were observed. In vivo, tea made with dried juvenile (DJ) leaves displays a peculiar behavior, meaning that some components may help simalikalactone D delivery or may be active in vivo only, therefore enhancing the expected curative effect of the traditional preparation.

From Tonic-cups to Bitter-cups: Kwasi bita beker from Suriname Determination, past and present use of an ancient galenic artefact.

In the main markets of Paramaribo (Suriname), many stands offer what is locally called "Bitter-cups", or "Kwasi bita beker", small footed-cups, roughly carved from a whitish wood. The use of these cups is strictly medicinal and it seems to be restricted to Suriname, as they are not found in neighbouring countries (Guyana, French Guiana). The aim of this study was to identify the botanical origin of Bitter-cups still in use in the Saramaka traditional medicine (as information from field people was controversial), and document the ethnopharmacology of this original galenical artefact. Microscopic and high performance liquid chromatography (HPLC) analyses were carried out on Bitter-cup, and anatomical criteria (marginal parenchyma band, size of intervessel and vessel-ray pits, rays width and rays composition, vessels clustering, frequency and size of parenchyma pits) together with HPLC profiles of the macerates showed that the wood cup was similar to Quassia amara L. (Simaroubaceae) wood. Ethnopharmacological investigation indicates that the use of these cups is simply due to the pharmacological properties attributed to "bitters", and is strongly linked to tradition and cultural attitudes. This study also emphasizes the long lasting use of these cups, now restricted to Suriname only, with almost no variation over one century.

Evaluation of the leishmanicidal activity of plants used by Peruvian Chayahuita ethnic group.

AIM OF THE STUDY: A total of 27 ethanolic plant extracts from 27 species were screened for leishmanicidal activity in vitro against Leishmania amazonensis. Most of the selected species (19) are traditionally used by the Chayahuitas, an Amazonian Peruvian ethnic group, to treat skin affections and/or leishmaniasis. MATERIAL AND METHODS: A colorimetric method based on the reduction of tetrazolium salt (MTT) was used to measure the viability of Leishmania amazonensis promastigote and amastigote stages. RESULTS AND CONCLUSIONS: Only the leaves of two species of the Piperaceae family (Piper hispidum Sw., and Piper strigosum Trel.) showed good leishmanicidal activities (IC(50)<10 microg/ml against amastigotes). Roots of Tabernaemontana sananho Ruiz & Pav. (Apocynaceae), together with bark of Vismia tomentosa Ruiz & Pav. (Clusiaceae), fruits of Solanum straminifolium var straminifolium Jacq. (Solanaceae), and stems of Zamia lindenii Regel ex André (Cycadaceae) showed low activity against amastigote stage (IC(50) around 50 microg/ml). Of those only Tabernaemontana sananho displayed also good activity on promastigotes (IC(50)<10 microg/ml). Results are discussed herein, in relation with the traditional use of the plants and compared with other data from the relevant literature.

Blood schizontocidal activity of methylene blue in combination with antimalarials against Plasmodium falciparum.

Methylene blue (MB) is the oldest synthetic antimalarial. It is not used anymore as antimalarial but should be reconsidered. For this purpose we have measured its impact on both chloroquine sensitive and resistant Plasmodium strains. We showed that around 5 nM of MB were able to inhibit 50% of the parasite growth in vitro and that late rings and early trophozoites were the most sensitive stages; while early rings, late trophozoites and schizonts were less sensitive. Drug interaction study following fractional inhibitory concentrations (FIC) method showed antagonism with amodiaquine, atovaquone, doxycycline, pyrimethamine; additivity with artemether, chloroquine, mefloquine, primaquine and synergy with quinine. These results confirmed the interest of MB that could be integrated in a new low cost antimalarial combination therapy.

Antimalarial activity of some Colombian medicinal plants.

Antimalarial activity of 10 vegetal extracts (9 ethanolic extracts and 1 crude alkaloid extract), obtained from eight species traditionally used in Colombia to treat malaria symptoms, was evaluated in culture using Plasmodium falciparum chloroquine resistant (FcB2) strain and in vivo on rodent malaria Plasmodium berghei. The activity on ferriprotoporphyrin biomineralization inhibition test (FBIT) was also assessed. Against Plasmodium falciparum, eight extracts displayed good activity Abuta grandifolia (Mart.) Sandwith (Menispermaceae) leaves, Acacia farnesiana (L.) Willd. (Mimosaceae) leaves, Acnistus arborescens (L.) Schltdl. (Solanaceae) aerial part, Croton leptostachyus Kunth (Euphorbiaceae) aerial part, Piper cumanense Kunth (Piperaceae) fruits and leaves, Piper holtonii C. DC. (Piperaceae) aerial part and Xylopia aromatica (Lam.) Mart. (Annonaceae) bark with IC(50) values ranging from <1 to 2.1 microg/ml, while in the in vivo model only Abuta grandifolia alkaloid crude extract exhibits activity, inhibiting 66% of the parasite growth at 250 mg/kg/day. In the FBIT model, five extracts were active (Abuta grandifolia, Croton leptostachyus, Piper cumanense fruit and leaves and Xylopia aromatica).

Simalikalactone D is responsible for the antimalarial properties of an Amazonian traditional remedy made with Quassia amara L. (Simaroubaceae).

French Guiana (North-East Amazonia) records high malaria incidence rates. The traditional antimalarial remedy most widespread there is a simple tea made out from Quassia amara L. leaves (Simaroubaceae). This herbal tea displays an excellent antimalarial activity both in vitro and in vivo. A known quassinoid, simalikalactone D (SkD), was identified as the active compound, with an IC(50) value of 10nM against FcB1 Plasmodium falciparum chloroquine resistant strain in vitro. Lastly, it inhibits 50% of Plasmodium yoelii yoelii rodent malaria parasite at 3.7 mg/kg/day in vivo by oral route. These findings confirm the traditional use of this herbal tea.

Antimalarial remedies in French Guiana: a knowledge attitudes and practices study.

A "knowledge attitudes and practices" study about malaria treatments was undertaken in French Guiana, along with an ethnopharmacological study. One hundred and seventeen people from five different groups and nationalities (Creole, Palikur, Galibi, Brazilian, and European) answered the questionnaire. The results were analysed using univariate and multivariate statistical analysis. First, we evaluated the overall knowledge about malaria from the interviewed people. According to bio-medical concepts, we noticed that they have a good knowledge of this illness. Secondly, we studied the treatment used by sick people during their last malaria attack. We demonstrated that, although bio-medical treatment is available in this area, people use both modern drugs and traditional remedies. Finally, preventive attitudes have been examined. One-third of the interviewed people drink regularly some herbal remedy to prevent febrile illnesses and malaria, thus displaying a strong concern about this disease. The ethnopharmacological study highlighted the frequent use of traditional remedies, along with their mode of preparation and administration. A total of 34 different species (both from flora and fauna) have been registered as antimalarial. Twenty-seven are used for curative purposes, 20 as preventive and 13 of them are used for both purposes. Quassia amara (Simaroubaceae) whose antimalarial activity has already been demonstrated was the species most frequently used as antimalarial for curative and preventive purposes.

Evaluation of French Guiana traditional antimalarial remedies.

In order to evaluate the antimalarial potential of traditional remedies used in French Guiana, 35 remedies were prepared in their traditional form and screened for blood schizonticidal activity in vitro on Plasmodium falciparum chloroquine re4sistant strain (W2). Some of these extracts were screened in vivo against Plasmodium yoelii rodent malaria. Ferriprotoporphyrin inhibition test was also performed. Four remedies, widely used among the population as preventives, were able to inhibit more than 50% of the parasite growth in vivo at around 100 mg/kg: Irlbachia alata (Gentiananceae), Picrolemma pseudocoffea (Simaroubaceae), Quassia amara (Simaroubaceae), Tinospora crispa (Menispermaceae) and Zanthoxylum rhoifolium (Rutaceae). Five remedies displayed an IC50 in vitro < 10 microg/ml: Picrolemma pseudocoffea, Pseudoxandra cuspidata (Annonaceae) and Quassia amara leaves and stem, together with a multi-ingredient recipe. Two remedies were more active than a Cinchona preparation on the ferriprotoporphyrin inhibition test: Picrolemma pseudocoffea and Quassia amara. We also showed that a traditional preventive remedy, made from Geissospermum argenteum bark macerated in rum, was able to impair the intrahepatic cycle of the parasite. For the first time, traditional remedies from French Guiana have been directly tested on malarial pharmacological assays and some have been shown to be active.

Mode of antimalarial effect of methylene blue and some of its analogues on Plasmodium falciparum in culture and their inhibition of P. vinckei petteri and P. yoelii nigeriensis in vivo.

The antimalarial action of methylene blue (MB) was first noted by Paul Ehrlich in the late 19th century. Although it has only sporadically been adopted as a serviceable drug, the resolution of its antimalarial action seems warranted, as it is currently used for the treatment of various methemoglobinemias. In this work we have used MB, and its analogues Azures A (AZA), B (AZB), C (AZC), and thionin (TH), as well as the oxazine Celestine blue (CB) and azine Phenosaphranin (PS). All MB analogues inhibit the growth of various strains of Plasmodium falciparum in culture with IC50s in the 2 x 10(-9)-1 x 10(-7) M range, with the rank order MB approximately AZA > AZB > AZC > TH > PS > CB. The IC50s for a mammalian cell line were in the 3 x 10(-6)-4 x 10(-5) M range, and the rank order was TH approximately AZB > AZA approximately PS > AZC approximately CB > MB. As MB could affect cell growth through the oxidation of NADPH, we tested the action of the various compounds on the hexose-monophosphate shunt activity. Appreciable activation of the shunt was observed at 1 x 10(-5) M in both cell types, thus accounting for inhibition of growth of mammalian cells but not of parasites. All compounds were found to complex with heme in a rank order similar to their antimalarial effect. It is therefore suggested that MB and its congeners act by preventing the polymerization of heme, which is produced during the digestion of host cell cytosol in the parasite food vacuole, into hemozoin. In this respect, these compounds seem to act similarly to the 4-aminoquinoline antimalarials. All compounds effectively suppressed the growth of P. vinckei petteri in vivo with IC50 in the 1.2-5.2 mg/kg range, and MB and AZB suppressed P. yoelii nigeriensis in the 9-11 mg/kg range (i.e. at doses similar to those of chloroquine). The potential toxicity of these compounds may restrict their clinical use, but their impressive antimalarial activities suggest that the phenothiazine structure could serve as a lead compound for further drug development.

Antimalarial properties of soy-bean fat emulsions.

Intralipid and Ivelip are commercial preparations of soy-bean lipid extracts used for intravenous supplementation of lipids in various clinical conditions. They were found to inhibit the growth of Plasmodium falciparum in culture with an IC50 of 8.07 +/- 2.13 and 13.32 +/- 2.05 mg.ml-1, respectively. Intralipid rapidly and efficiently inhibited nucleic acid synthesis in cultured P. falciparum, exhibiting full inhibitory activity in less than 2 h. Ivelip injected intraperitoneally, was found by the 4-day suppressive test to be active in vivo against P. vinckei petteri within the normal recommended regimen for dietary lipid supply (0.5-4 g.kg-1), but it was impossible to obtain a radical cure even with very high doses (6.4 g.kg-1). Ivelip was less effective against P. berghei and P. yoelii nigeriensis. As Ivelip showed no interference with the antimalarial activity of chloroquine, it could be considered for use in the treatment of severe human malaria in association with 4-aminoquinolines to expedite the clearance of parasites.

Comparative pharmacology of low molecular weight heparins: implications of manufacturing processes on biological effects.

With the recent development of numerous low molecular weight heparins (LMWHs), a certain amount of concern has become evident as to the equivalency of each agent. In a comprehensive study, we have taken the seven available LMWHs to directly compare their in vitro and in vivo (subcutaneous) antithrombotic properties in one laboratory setting. Where possible, various batches of one LMWH were evaluated. Our findings were that variations of in vivo activity were observed between the LMWHs studied. Some activities were significantly different from placebo, whereas others were not. Depending on the assay chosen significant differences could also be observed for the in vitro activity.

Antithrombotic activity of recombinant hirudin in the rat: a comparative study with heparin.

Hirudin, a potent inhibitor of blood coagulation, differs in its antithrombotic activity according to the source of isolation. It was therefore of interest to study recombinant hirudin. Hirudin was obtained by a genetic process from E. coli. Its antithrombotic action was investigated in an experimental (rat) model of venous thrombosis and was compared to heparin whose results are known. Heparin (400 micrograms/kg) and hirudin (12.5, 25 and 50 micrograms/kg) present an antithrombotic effect and limit the extension of an existing thrombus (p less than 0.05). Higher heparin dosages increase the bleeding time mean value (p less than 0.05) whereas hirudin does not. So, recombinant hirudin presents the same antithrombotic action as heparin but with very inferior dosage. This activity seems not dose-dependent and is associated to weak hemorrhagic effects.

Submicron oil-in-water emulsion formulations for mefloquine and halofantrine: effect of electric-charge inducers on antimalarial activity in mice.

Stearylamine, oleic acid, phosphatidylserine and dicetylphosphate have been studied to determine their capacity to induce electric charge on non-ionic submicron emulsions containing halofantrine and mefloquine. The in-vivo antimalarial activity of drug-loaded emulsions, evaluated in mice, was affected by the nature of the additives used. In particular, the electric-charge inducers markedly affected the pharmacological activity of mefloquine, but not of halofantrine. After subcutaneous administration ED50 values (the doses affording 50% protection) were 3 and 15 mg kg(-1), respectively, for halofantrine and mefloquine emulsions without charge inducers. The mefloquine-loaded emulsions with charge inducers were active at 10 mg kg(-1) for dicetylphosphate, 17 mg kg(-1) for phosphatidylserine, 23 mg kg(-1) for oleic acid and 27 mg kg(-1) for stearylamine, again after subcutaneous administration. This work has enabled the formulation of stable emulsions, incorporating drugs with high antimalarial activity, which are proposed for parenteral delivery of these fairly soluble drugs.

A search for natural bioactive compounds in Bolivia through a multidisciplinary approach. Part VI. Evaluation of the antimalarial activity of plants used by Isoceño-Guaraní Indians.

Seventy-seven plant extracts (corresponding to 62 different species) traditionally used by the Isoceño-Guaraní, a native community living in the Bolivian Chaco, were screened for antimalarial activity in vitro on Plasmodium falciparum chloroquine sensitive strain (F32), and on ferriprotoporphyrin (FP) IX biocrystallisation inhibition test (FBIT). Among these extracts, seven displayed strong in vitro antimalarial activity, and 25 were active in the FBIT test. Positive results on both tests were recorded for six extracts: Argemone subfusiformis aerial part, Aspidosperma quebracho-blanco bark, Castela coccinea leaves and bark, Solanum argentinum leaves and Vallesia glabra bark. Results are discussed in relation with Isoceño-Guaraní traditional medicine. Further studies to be undertaken in relation with these results are also highlighted.

A search for natural bioactive compounds in Bolivia through a multidisciplinary approach. Part III. Evaluation Of the antimalarial activity of plants used by Alteños Indians.

A total of 40 plant extracts traditionally used by the Alteños Indians, a native community living between the Andean block and the tropical valleys of Bolivia, were screened for antimalarial activity in vitro on Plasmodium falciparum chloroquine resistant (Indo) strain, and in vivo on rodent malaria Plasmodium vinckei petteri. Eleven extracts displayed good or moderate activity in vivo, and ten extracts good or very good antimalarial activity in vitro. Results of the screening are discussed here, in relation with the traditional use of plants.

A search for natural bioactive compounds in Bolivia through a multidisciplinary approach. Part IV. Is a new haem polymerisation inhibition test pertinent for the detection of antimalarial natural products?

The search for new antimalarial agents in plant crude extracts using traditional screening tests is time-consuming and expensive. New in vitro alternative techniques, based on specific metabolic or enzymatic process, have recently been developed to circumvent testing of antimalarial activity in parasite culture. The haem polymerisation inhibition test (HPIA) was proposed as a possible routine in vitro assay for the detection of antimalarial activity in natural products. A total of 178 plant extracts from the Pharmacopeia of the Bolivian ethnia Tacana, were screened for their ability to inhibit the polymerisation of haematin. Five extracts from Aloysia virgata (Ruíz & Pavón) A.L. Jussieu (Verbenaceae), Bixa orellana L. (Bixaceae), Caesalpinia pluviosa D.C. (Caesalpiniaceae), Mascagnia stannea (Griseb) Nied. (Malpighiaceae) and Trichilia pleenea (Adr. Jussieu) (Meliaceae) demonstrated more than 70% inhibition of haematin polymerisation at 2.5 mg/ml. The extracts were also tested for antimalarial activity in culture against F32 strain (chloroquine-sensitive) and D2 strain (chloroquine-resistant) of Plasmodium falciparum and in vivo against P. berghei. The extract from Caesalpinia pluviosa was the only one that showed activity in HPIA and in the classical test in culture. The accuracy and pertinence of HPIA, applied to natural products is discussed.