Trypanosomiasis and leishmaniasis are important parasitic diseases affecting millions of people in Africa, Asia, and South America. In a previous study, we identified several flavonoid glycosides as antiprotozoal principles from a Turkish plant. Here we surveyed a large set of flavonoid aglycones and glycosides, as well as a panel of other related compounds of phenolic and phenylpropanoid nature, for their in vitro activities against Trypanosoma brucei rhodesiense , Trypanosoma cruzi , and Leishmania donovani . The cytotoxicities of more than 100 compounds for mammalian L6 cells were also assessed and compared to their antiparasitic activities. Several compounds were investigated in vivo for their antileishmanial and antitrypanosomal efficacies in mouse models. Overall, the best in vitro trypanocidal activity for T. brucei rhodesiense was exerted by 7,8-dihydroxyflavone (50% inhibitory concentration [IC ₅₀ ], 68 ng/ml), followed by 3-hydroxyflavone, rhamnetin, and 7,8,3′,4′-tetrahydroxyflavone (IC ₅₀ s, 0.5 μg/ml) and catechol (IC ₅₀ , 0.8 μg/ml). The activity against T. cruzi was moderate, and only chrysin dimethylether and 3-hydroxydaidzein had IC ₅₀ s less than 5.0 μg/ml. The majority of the metabolites tested possessed remarkable leishmanicidal potential. Fisetin, 3-hydroxyflavone, luteolin, and quercetin were the most potent, giving IC ₅₀ s of 0.6, 0.7, 0.8, and 1.0 μg/ml, respectively. 7,8-Dihydroxyflavone and quercetin appeared to ameliorate parasitic infections in mouse models. Generally, the test compounds lacked cytotoxicity in vitro and in vivo. By screening a large number of flavonoids and analogues, we were able to establish some general trends with respect to the structure-activity relationship, but it was not possible to draw clear and detailed quantitative structure-activity relationships for any of the bioactivities by two different approaches. However, our results can help in directing the rational design of 7,8-dihydroxyflavone and quercetin derivatives as potent and effective antiprotozoal agents.

The sesquiterpene lactone helenalin is a potent anti-inflammatory drug whose molecular mechanism of action remains unclear despite numerous investigations. We have previously shown that helenalin and other sesquiterpene lactones selectively inhibit activation of the transcription factor NF-kappaB, a central mediator of the human immune response. These drugs must target a central step in NF-kappaB pathway, since they inhibit NF-kappaB induction by four different stimuli. It has previously been reported that sesquiterpene lactones exert their effect by inhibiting degradation of IkappaB, the inhibitory subunit of NF-kappaB. These data contradicted our report that IkappaB is not detectable in helenalin-treated, ocadaic acid-stimulated cells. Here we use confocal laser scanning microscopy to demonstrate the presence of IkappaB-released, nuclear NF-kappaB in helenalin-treated, tumor necrosis factor-alpha stimulated cells. These data show that neither IkappaB degradation nor NF-kappaB nuclear translocation are inhibited by helenalin. Rather, we provide evidence that helenalin selectively alkylates the p65 subunit of NF-kappaB. This sesquiterpene lactone is the first anti-inflammatory agent shown to exert its effect by directly modifying NF-kappaB.

Sesquiterpene lactones (SLs) have potent anti-inflammatory properties. We have shown previously that they exert this effect in part by inhibiting activation of the transcription factor NF-κB, a central regulator of the immune response. We have proposed a molecular mechanism for this inhibition based on computer molecular modeling data. In this model, SLs directly alkylate the p65 subunit of NF-κB, thereby inhibiting DNA binding. Nevertheless, an experimental evidence for the proposed mechanism was lacking. Moreover, based on experiments using the SL parthenolide, an alternative mode of action has been proposed by other authors in which SLs inhibit IκB-α degradation. Here we report the construction of p65/NF-κB point mutants that lack the cysteine residues alkylated by SLs in our model. In contrast to wild type p65, DNA-binding of the Cys³⁸ → Ser and Cys^38,120 → Ser mutants is no longer inhibited by SLs. In addition, we provide evidence that parthenolide uses a similar mechanism to other SLs in inhibiting NF-κB. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-κB most probably by alkylating p65 at Cys³⁸. Although a slight inhibition of IκB degradation was detected for all SLs, the amount of remaining IκB was too low to explain the observed NF-κB inhibition.

Alcoholic extracts prepared form Arnicae flos, the collective name for flowerheads from Arnica montana and A. chamissonis ssp. foliosa, are used therapeutically as anti-inflammatory remedies. The active ingredients mediating the pharmacological effect are mainly sesquiterpene lactones, such as helenalin, 11alpha,13-dihydrohelenalin, chamissonolid and their ester derivatives. While these compounds affect various cellular processes, current data do not fully explain how sesquiterpene lactones exert their anti-inflammatory effect. We show here that helenalin, and, to a much lesser degree, 11alpha,13-dihydrohelenalin and chamissonolid, inhibit activation of transcription factor NF-kappaB. This difference in efficacy, which correlates with the compounds' anti-inflammatory potency in vivo, may be explained by differences in structure and conformation. NF-kappaB, which resides in an inactive, cytoplasmic complex in unstimulated cells, is activated by phosphorylation and degradation of its inhibitory subunit, IkappaB. Helenalin inhibits NF-kappaB activation in response to four different stimuli in T-cells, B-cells and epithelial cells and abrogates kappaB-driven gene expression. This inhibition is selective, as the activity of four other transcription factors, Oct-1, TBP, Sp1 and STAT 5 was not affected. We show that inhibition is not due to a direct modification of the active NF-kappaB heterodimer. Rather, helenalin modifies the NF-kappaB/IkappaB complex, preventing the release of IkappaB. These data suggest a molecular mechanism for the anti-inflammatory effect of sesquiterpene lactones, which differs from that of other nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and acetyl salicylic acid.

Infections with protozoan parasites are a major cause of disease and mortality in many tropical countries of the world. Diseases caused by species of the genera Trypanosoma (Human African Trypanosomiasis and Chagas Disease) and Leishmania (various forms of Leishmaniasis) are among the seventeen “Neglected Tropical Diseases” (NTDs) defined as such by WHO due to the neglect of financial investment into research and development of new drugs by a large part of pharmaceutical industry and neglect of public awareness in high income countries. Another major tropical protozoan disease is malaria (caused by various Plasmodium species), which -although not mentioned currently by the WHO as a neglected disease- still represents a major problem, especially to people living under poor circumstances in tropical countries. Malaria causes by far the highest number of deaths of all protozoan infections and is often (as in this review) included in the NTDs. The mentioned diseases threaten many millions of lives world-wide and they are mostly associated with poor socioeconomic and hygienic environment. Existing therapies suffer from various shortcomings, namely, a high degree of toxicity and unwanted effects, lack of availability and/or problematic application under the life conditions of affected populations. Development of new, safe and affordable drugs is therefore an urgent need. Nature has provided an innumerable number of drugs for the treatment of many serious diseases. Among the natural sources for new bioactive chemicals, plants are still predominant. Their secondary metabolism yields an immeasurable wealth of chemical structures which has been and will continue to be a source of new drugs, directly in their native form and after optimization by synthetic medicinal chemistry. The current review, published in two parts, attempts to give an overview on the potential of such plant-derived natural products as antiprotozoal leads and/or drugs in the fight against NTDs.