Cytotoxic activity of some phenanthroindolizidine N-oxide alkaloids from Cynanchum vincetoxicum.

Two previously known phenanthroindolizidine alkaloids, (-)-10beta-antofine N-oxide (1) and (-)-10beta, 13aalpha-14beta-hydroxyantofine N-oxide (2), and a novel alkaloid, (-)-10beta,13aalpha-secoantofine N-oxide (3), were isolated from aerial parts of Cynanchum vincetoxicum. Their structures were established by means of NMR methods, including COSY, NOESY, HSQC, and HMBC experiments, as well as from their CD spectra. Cytotoxic activity of the alkaloids was assessed in vitro using both a drug-sensitive KB-3-1 and a multi-drug-resistant KB-V1 cancer cell line. The antofine derivatives (1 and 2) showed pronounced cytotoxicity against the drug-sensitive cell line (IC(50) values about 100 nM), whereas the secoantofine derivative (3) was considerably less active. The KB-V1 cell line showed a marginal resistance against all alkaloids, demonstrating that these compounds are poor substrates for the P-glycoprotein (P-170) efflux pump.

Leishmanicidal, antiplasmodial and cytotoxic activity of indole alkaloids from Corynanthe pachyceras.

Five indole alkaloids, corynantheidine, corynantheine, dihydrocorynantheine, alpha-yohimbine and corynanthine were isolated from bark of Corynanthe pachyceras K. Schum. (Rubiaceae). The structures were established by spectroscopic methods, including previously unreported assignment of all 1H-NMR resonances by COSY and NOESY experiments. These and related alkaloids showed pronounced activity against Leishmania major promastigotes (IC50 at the micromolar level) but no significant in vitro antiplasmodial activity (against chloroquine-sensitive Plasmodium falciparum). Cytotoxicity assessed with drug sensitive KB-3-1 and multidrug-resistant KB-V1 cell lines was low; the alkaloids are apparently not substrates for the P-glycoprotein (P-170) efflux pump.

Biodegradability of the molluscicidal saponins of Phytolacca dodecandra.

The biodegradability of water-extracted saponins of berries from the Endod plant, Phytolacca dodecandra L'Herit, was evaluated under OECD standardized conditions. Persistence of the saponins was evaluated by determination of saponin concentrations in water over a 30-day period, using a quantitative HPLC method and a semiquantitative hemolytic assay, which is considered a potential field method. The two methods were compared. Bioassays were simultaneously conducted using Biomphalaria glabrata fresh water snails to assess the molluscicidal potency over time. All experiments were carried out in water, which was chemically balanced to sustain snails, and to one set river water was added to the medium to mimic natural conditions. (1) Saponin concentrations in water treated with an aqueous extract of P. dodecandra cultivar E44 were stable for 2 days then rapidly decreased during the third and fourth day. Lethal concentrations to snails were LC50 = 9.6 mg/l (95% CI: 6.3 to 19.4) in one set and LC50 = 6.8 mg/l (95% CI: 5.4 to 12.2) in the other. Adding river water to one set of the experiments had no effect on the mean saponin concentration over time (F = 0.02, P = >0.05). (2) Comparison of the hemolytic assay with the HPLC method showed no significant difference in mean saponin concentrations (t = 0.32, P > 0.05, 95% CI: -2.67 to 3.64), and a correlation of r2 = 0.88 between the two methods. (3) The saponin fraction of an aqueous extract of P. dodecandra was readily biodegraded (t1/2 = 15.8 h), and the complete consumption within a 10-day window indicates ready degradability in aquatic environments under aerobic conditions. (4) These results show that the use of Endod berries for snail control in schistosomiasis-infested water bodies is environmentally acceptable.

Structure-activity studies: in vitro antileishmanial and antimalarial activities of anthraquinones from Morinda lucida.

Anthraquinones have been isolated by bioassay-guided fractionation from Morinda lucida. Structure-activity studies show that an aldehyde group at C-2 and a phenolic hydroxy group at C-3 enhance the activity of the anthraquinones against the growth of Plasmodium falciparum and promastigotes of Leishmania major in vitro.

Antiprotozoal properties of 16,17-dihydrobrachycalyxolide from Vernonia brachycalyx.

Extracts of the leaves from Vernonia brachycalyx showed in vitro activity against Plasmodium falciparum and promastigotes of Leishmania major. The germacrane dilactone 16,17-dihydrobrachycalyxolide (1) which was previously isolated from the aerial parts of the plant was shown to be the major antiplasmodial principle. An X-ray crystallographic analysis established the absolute configuration and some signals in the NMR spectra were reassigned. 16,17-Dihydrobrachycalyxolide (1) elicited a strong antiplasmodial and antileishmanial activity but also a high toxicity against human lymphocytes.

Alkamides from Phyllanthus fraternus.

Two alkamides E,E-2,4-octadienamide and E,Z-2,4-decadienamide have been isolated from Phyllanthus fraternus, a plant used in Ghanaian traditional medicine to treat malaria. The compounds possess an alpha, beta, gamma, delta-unsaturated conjugated amide, a feature believed to enhance antiplasmodial activity. By means of an in vitro assay the two alkamides have been shown to possess moderate antiplasmodial activity.

Antiprotozoal compounds from Asparagus africanus.

Two antiprotozoal compounds have been isolated from the roots of Asparagus africanus Lam. (Liliaceae), a new sapogenin, 2 beta, 12 alpha-dihydroxy-(25R)-spirosta-4,7-dien-3-one (1), which was named muzanzagenin, and the lignan (+)-nyasol (2), (Z)-(+)-4,4'-(3-ethenyl-1-propene-1,3-diyl)-bisphenol. The structure of the sapogenin was elucidated by MS and by 1D and 2D NMR methods and established by a single crystal X-ray analysis. (+)-Nyasol potently inhibits the growth of Leishmania major promastigotes, the IC50 being 12 microM, and moderately inhibits Plasmodium falciparum schizonts with the IC50 49 microM. These concentrations only moderately affect the proliferation of human lymphocytes. Muzanzagenin showed a moderate in vitro activity in all three tests, the IC50 against leishmania promastigotes was 70 microM, and against four different malaria schizont strains the IC50 values were 16, 163, 23, and 16 microM, respectively.

Two new antiprotozoal 5-methylcoumarins from Vernonia brachycalyx.

Two new isomeric 5-methylcoumarins, 2'-epicycloisobrachycoumarinone epoxide (1) and cycloisobrachycoumarinone epoxide (2), have been isolated from the roots of Vernonia brachycalyx by means of bioactivity-guided fractionation. The structures were elucidated by MS and NMR spectroscopic methods. Compounds 1 and 2 showed in vitro activity against Leishmania major promastigotes and against Plasmodium falciparum schizonts and demonstrated an inhibition on the proliferation of human lymphocytes, which was significantly weaker than the antiparasitic effects.

Molluscicidal saponins from Catunaregam nilotica.

Two new saponins were isolated from the fruits of Catunaregam nilotica Stapf, syn. Lachnosiphonium nilotica; Randia nilotica; Xeromphis nilotica. Their structures were determined mainly by spectroscopic methods as 3- O-[O-alpha-L-rhamnopyranosyl-(1-->3)-O-[O-beta-D-glucopyranosyl-(1 -->3)]- beta-D-glucopyranosyl]oleanolic acid and 28-O-beta-D- glucopyranosyl-3-O-[O-alpha-L-rhamnopyranosyl-(1-->3)-O[O-beta-D- glucopyranosyl]-beta-D-glycopyranosyl]oleanolate. The monodesmosidic saponin is a potent molluscicide against the schistosomiasis transmitting snail Biomphalaria glabrata with a LC50 value of 3 ppm. In addition two known saponins, 3-O-[2', 3'-di-O-(beta-D-glucopyranosyl)-beta-D- glucopyranosyl]oleanolic acid and 3-O-[O-beta-D-glucopyranosyl(1-->3)- beta-D-glucopyranosyl]oleanolic acid, were identified and their molluscicidal activity determined, the LC50 values being 26 and 3 ppm, respectively. Initial molluscicidal screening of the crude water and ethanol extracts revealed 100% snail mortality at concentrations of 100 and 50 ppm, respectively. The haemolytic activity of the molluscicidal saponins was determined as well and the HC50 values towards bovine erythrocytes found to be 3 ppm for the new saponin, and 16 and 2 ppm, respectively, for the two known saponins.

Molluscicidal saponins from a Zimbabwean strain of Phytolacca dodecandra.

Three new monodesmosidic saponins, all glycosides of 2 beta-hydroxyoleanolic acid, were isolated from an aqueous extract of a Zimbabwean strain of Phytolacca dodecandra. Their structures were, mainly by spectroscopic methods (LSIMS, 1H NMR, COSY, NOESY, TOCSY, J-resolved 1H NMR, 13C NMR, HETCOR), established as 3-O-[2',4'-di-O-(beta-D-glucopyranosyl)-beta-D-glucopyranosyl]2 beta-hydroxyoleanolic acid, 3-O-[O-alpha-L-rhamnopyranosyl-(1-->2)-O-[beta-D-galactopyranosyl- (1-->3)]-beta-D-glucopyranosyl]2 beta-hydroxyoleanolic acid and 3-O-[3'-O-(beta-D-galactopyranosyl)-beta-D-glucopyranosyl]2 beta-hydroxyoleanolic acid. Two of the saponins were submitted to a preliminary screening for molluscicidal activity against the schistosomiasis transmitting snail Biomphalaria glabrata and showed, respectively, strong and weak activity. In addition, four saponins previously reported from other strains of Phytolacca dodecandra were identified.

Investigation of the diurnal, ontogenetic and seasonal variation in the molluscicidal saponin content of Phytolacca dodecandra aqueous berry extracts.

Daily variation in the content of the saponins of the berries of Phytolacca dodecandra was determined in berries harvested at the full grown green unripe stage of development once every two hours. The saponin content was quantitatively determined by HPLC analysis of the molluscicidal monodesmosidic saponins in the aqueous extract of the crushed berries. No significant daily variations were observed in extracts of berries collected at different times of day (P > 0.05 by ANOVA). But, seasonal variation was apparent in extracts prepared from berries harvested from the same plants once every two weeks. Berries of highest saponin content were those harvested during the dry season and just before the onset of the rainy season. The seasonal variation was attributed to increased water stress during the dry season. Very young racemes had the highest saponin content, while mature berries produced the lowest saponin concentration. There was, however, consistency in the berry saponin composition during the day, season and all stages of berry development.

Licochalcone A, a novel antiparasitic agent with potent activity against human pathogenic protozoan species of Leishmania.

Licochalcone A, an oxygenated chalcone isolated from the roots of Chinese licorice plant, inhibited the growth of both Leishmania major and Leishmania donovani promastigotes and amastigotes. The structure of the licochalcone A was established by mass and nuclear magnetic resonance spectroscopies and by synthesis, and its purity was verified by high-pressure liquid chromatography. The 50% inhibition of growth of logarithmic- and stationary-phase promastigotes of L. major, as measured by [3H]thymidine uptake, were 4 and 2.5 micrograms/ml, respectively. The growth of L. major promastigotes was totally inhibited after a 20-h incubation period with licochalcone A at 5 micrograms/ml. At a concentration of 0.5 microgram/ml, licochalcone A markedly reduced the infection rate of human peripheral blood monocyte-derived macrophages and U937 cells with L. major promastigotes and exhibited a strong intracellular killing of the parasite. These data show that intracellular Leishmania amastigotes are more susceptible than promastigotes to licochalcone A. Results of studies on the site of action of licochalcone A indicate that the target organelle appears to be the parasite mitochondria. These findings demonstrate that licochalcone A in concentrations that are nontoxic to host cells exhibits a strong antileishmanial activity and that appropriate substituted chalcones might be a new class of antileishmanial drugs.

The molluscicidal activity of coumarins from Ethulia conyzoides and of dicumarol.

The molluscicidal principles of Ethulia conyzoides were identified as ethuliacoumarin A (1) and isoethuliacoumarin A (2). Ethuliacoumarin A possessed an LC90 between 19 and 23.5 ppm depending on the age of the snail against Biomphalaria glabrata, and between 12 and 15 ppm against Bulinus truncatus. In addition, ethuliacoumarin A was found to be cercaricidal at 25 ppm and ovicidal. Ethuliacoumarin has the structural requirements considered essential for anticoagulant activity. Consequently the anticoagulant dicumarol (4) was tested and found to be molluscicidal in the range from 2.5 to 10 ppm. In contrast, the coumarin anticoagulant warfarin (3) did not show molluscicidal activity.

Endod: safety evaluation of a plant molluscicide.

Toxicity tests, in accordance with the Minimal Data Requirements (Tier 1) of the OECD Guidelines for Pre-Market Chemicals, were conducted on a standard extract (Endod-S) from the unripe berries of Phytolacca dodecandra, a potent botanical molluscicide of potential importance in the control of schistosomiasis. In acute mammalian toxicity tests, except for the eye irritation toxicity test which indicated severe irritancy, all test results were classified as either nontoxic or slightly toxic. Eye protection is therefore recommended during berry crushing and handling of dry powders. Ecotoxicity tests indicated that Endod is no more toxic than currently recommended synthetic molluscicides; however, environmental fate and additional local ecotoxicity tests are recommended for nontarget aquatic organisms present in the endemic situations of field trials. Given these toxicological data and recognizing the need for an affordable, locally cultivated, botanical molluscicide, it is concluded that field trials of Endod in schistosomiasis control are now justifiable.