Geoffrey C. Kirby

In vitro and In vivo evaluation of betulinic acid as an antimalarial

The lupane‐type triterpene betulinic acid was isolated from an ethanol extract of the root bark of the Tanzanian tree Uapaca nitida Müll‐Arg. (Euphorbiaceæ). The in vitro antiplasmodial IC50 values of betulinic acid against chloroquine resistant (K1) and sensitive (T9‐96) Plasmodium falciparum were found to be 19.6 µg/mL and 25.9 µg/mL, respectively. The in vitro activities of several related triterpenes were also evaluated. Betulin was found to be inactive at 500 µg/mL for both K1 and T9‐96. Ursolic acid exhibited IC50 values of 36.5 µg/mL and 28 µg/mL, and oleanolic acid exhibited IC50 values of 88.8 µg/mL and 70.6 µg/mL against K1 and T9‐96, respectively. When betulinic acid was tested for in vivo activity in a murine malaria model (P. berghei) the top dosage employed of 250 mg/kg/day was ineffective at reducing parasitaemia and exhibited some toxicity. Betulinic acid has not previously been evaluated for in vivo activity. This is believed to be the first compound to be isolated from U. nitida. Copyright

In vittro studies on the mode of action of quassinoids with activity against chloroquine-resistant Plasmodium falciparum

Using the incorporation of [3H]isoleucine or [3H]hypoxanthine into acid-insoluble products as indices of protein- and nucleic acid-synthetic activity, respectively, it was shown that seven plant-derived quassinoids with differing chemical substitutions all inhibited protein synthesis more rapidly than nucleic acid synthesis in human erythrocytes infected with Plasmodium falciparum, in vitro. Five quassinoids (ailanthinone, bruceantin, bruceine B, glaucarubinone and holacanthone) were effective within 30 min at doses 10 times their 48 hr in vitro IC50 values. Chaparrin and glaucarubol differed in that they did not inhibit protein synthesis during the time course of these experiments when applied at 10 times their in vitro IC50 values. When these compounds were used at 209 and 114 times their respective IC50 values, their observed effects were identical to those of the other quassinoids studied. The time (t50) at which nucleic acid synthesis was reduced to 50% of control was directly proportional to the t50 for protein synthesis, suggesting that failure of nucleic acid synthesis is a consequence of inhibition of protein synthesis. It is concluded that in the malaria parasite, as in eukaryote models, quassinoids are rapid and potent inhibitors of protein synthesis, and that this is most likely due to effects upon the ribosome, rather than upon nucleic acid metabolism.

Toward the chemical ecology of medicinal plant use in chimpanzees: The case ofVernonia amygdalina, a plant used by wild chimpanzees possibly for parasite-related diseases

The bitter and related constituents have been isolated fromVernonia amygdalina (Compositae), a plant ingested by wild chimpanzees possibly suffering from parasite-related diseases in the Mahale Mountains National Park, Tanzania. Isolated from the plant were four known sesquiterpene lactones, seven new steroid glucosides, and two aglycones of the glucosides. The sesquiterpene lactones showed significant in vitro antischistosomal, plasmodicidal, and leishmanicidal activities. Antischistosomal activity was also found for the major steroid glucoside, vernonioside B1. A trend in the glucosides to show significant antischistosomal, plasmodicidal, and amebicidal activities when the sugar moiety was removed, was observed. Vernodalin, judged as the most significant constituent for antiparasitic activities in vitro, was tested for in vivo antischistosomal effect. It was, however, highly toxic to the cercaria-infected mouse. Chimpanzees have been only rarely observed to ingest anything but the pith of the young stem. The occurrence of vernonioside B1 and its aglycone vernoniol B1, the major constituents among the steroid-related constituents, were detected at significant levels in the pith. However, vernodalin was abundant only in the leaves and bark. Thus, chimpanzees at Mahale were hypothesized to control parasite-related diseases by ingesting the young pith of this tree containing steroid-related constituents.

Medicinal plants and the control of protozoal disease, with particular reference to malaria

Malaria and other protozoal diseases continue to pose serious health problems world-wide. Resistance of the malaria parasites, Plasmodium spp., to drugs such as chloroquine (and, more lately, quinine) occurs with increasing frequency and underlies the necessity to develop new agents for malaria chemotherapy; in the case of diseases caused by species of Leishmania and Trypanosoma there has always been a marked paucity of effective drugs, particularly those with a wide safety margin and minimal or no undesirable side effects. Novel drugs, are required to help alleviate morbidity and mortality and to contribute to the world-wide control of these diseases, in part by helping to reduce the reservoirs of infection. Reliance upon plants for the treatment of disease is high in the developing world and such plants offer a source of new molecules. Research centered upon Plasmodium has produced a number of findings which now prompt the formulation of important questions which may influence and focus the direction of phytotherapy research in the future.

Antimalarial Activity of Cryptolepine and Some Other Anhydronium Bases

Eight naturally occurring anhydronium bases and the synthetic quaternary compound Nb‐methylharmalane were tested against Plasmodium falciparum (strain K1) in vitro. Cryptolepine was found to have similar activity to that of chloroquine but alstonine, 5,6‐dihydroflavopereirine, matadine, Nb‐methylharmalane, melinonine F, normelinonine F, strychnoxanthine and serpentine were found to have little activity. Cryptolepine, given orally to mice infected with Plasmodium berghei berghei was found to have moderate antimalarial activity; parasitaemia was suppressed by 80% at 50 mg/kg/day.

Activity of extracts and naphthylisoquinoline alkaloids from Triphyophyllum peltatum, Ancistrocladus abbreviatus and A. barteri against Plasmodium falciparum in vitro

Abstract Five extracts from the tropical plant species Triphyophyllum peltatum, Ancistrocladus abbreviatus and A. barteri, and six pure naphthylisoquinoline alkaloids derived from these species have been examined for their antiplasmodial activity. These species are well-known in the traditional medicine of West Africa and are used for the treatment of fevers, malaria and other diseases. The extracts and alkaloids were tested against the asexual erythrocytic stages of two strains of Plasmodium falciparum in vitro (K1/chloroquine-resistant and NF 54/64, clone A1A9/ chloroquine-sensitive). Incorporation of 3H-hypoxanthine was measured in the presence of the test substances after 42 hr of incubation at 37°. All extracts and three alkaloids displayed activity. The two most potent compounds were dioncopeltine A and dioncophylline B. Structure-activity considerations indicate two possible criteria for antiplasmodial activity: an R-configuration at C-3 associated with the absence of an oxygen substituent at C-6 and the absence of N-methylation.

Alstonia species: are they effective in malaria treatment?

A review of the literature on Alstonia species indicates that evidence in support of their effectiveness in the treatment of malaria is controversial. The antiprotozoal activity of the major alkaloid present in Alstonia species, echitamine, was assessed in vitro against Plasmodium falciparum and Giardia intestinalis. Echitamine displayed little antiplasmodial activity, but two quinoline alkaloids from A. coriacea (corialstonine and corialstonidine) were found to have some activity against P. falciparum although this was approximately 10 times less than that of quinine. None of the three Alstonia alkaloids was active against G. intestinalis. These results are discussed in the context of previously published data.

Comparison of the in vitro activities of quassinoids with activity against Plasmodium falciparum, anisomycin and some other inhibitors of eukaryotic protein synthesis.

Using the inhibition of incorporation of [3H]hypoxanthine as an index of viability of malaria parasites, it was shown that a chloroquine-sensitive strain of Plasmodium falciparum (T9-96) and a chloroquine-resistant strain (K1) did not differ in their sensitivities to the quassinoids ailanthinone, bruceantin and chaparrin. Similarly, there were no differences between the strains in their sensitivities to the protein synthesis inhibitors anisomycin, deacetylanisomycin, cephalotaxine, homoharringtonine, cycloheximide, puromycin and puromycin aminonucleoside. The IC50 values derived for ailanthinone and bruceantin, cycloheximide, homoharringtonine and puromycin were in the nanomolar range, whereas those for the anisomycins, cephalotaxine and the aminonucleoside of puromycin were micromolar or greater. Those drugs tested which contain an ester moiety (ailanthinone, bruceantin, anisomycin, homoharringtonine) were more active than the related drugs (chaparrin, deacetylanisomycin, cephalotaxine) that do not. Cross-resistance to inhibitors of protein synthesis appeared not to accompany resistance to chloroquine.

Quassinoids Exhibit Greater Selectivity Against Plasmodium Falciparum Than Against Entamoeba Histolytica, Giardia Intestinalis Or Toxoplasma Gondii In Vitro

ABSTRACT. The in vitro activities of a series of quassinoids against Plasmodium falciparum, Entamoeba histolytica, Giardia intestinalis and Toxoplasma gondii have been compared with their in vitro cytotoxic effects against KB cells (human epidermoid carcinoma of the nasopharynx). All of the compounds tested were more toxic to KB cells than to G. intestinalis, but four (ailanthinone, bruceine D, brusatol and glaucarubinone) were slightly less toxic to KB cells than to E. histolytica. Glaucarubinone was similarly more toxic to intracellular T. gondii than to KB cells but ailanthinone was more selective (36 times more toxic to T. gondii than to KB cells). All of the compounds were more toxic to P. falciparum than to KB cells; the most selective quassinoids—glaucarubinone, bruceine D, ailanthinone and brusatoi—were found to have toxicity/activity ratios of 285, 76, 48 and 32 respectively. These results suggest that quassinoids have a selective action on P. falciparum. Further studies to elucidate the basis for this are in progress.

Plasmodium falciparum: effects of phaeanthine, a naturally-occurring bisbenzylisoquinoline alkaloid, on chloroquine-resistant and -sensitive parasites in vitro, and its influence on chloroquine activity.

Phaeanthine, a bisbenzylisoquinoline alkaloid which occurs naturally in Triclisia species, was extracted from Triclisia patens (Menispermaceae) obtained from Sierra Leone (West Africa). In vitro, phaeanthine was found to be twice as potent against a chloroquine-resistant Plasmodium falciparum strain (K1), as against a chloroquine-sensitive clone (T9-96), with 50% inhibitory concentrations of 365.85 (+/- 11.41) nM and 704.87 (+/- 81.48) nM respectively. At a sub-inhibitory concentration of 80.35 nM, chloroquine resistance was not reversed by phaeanthine. Isobolograms constructed from experiments with chloroquine/phaeanthine combinations showed antagonism in T9-96 and an additive effect in K1. In a 48-hour microtest, phaeanthine at antimalarial concentrations showed no cytotoxicity to mammalian (KB) cells in vitro.