Characterization of noncovalent complexes of antimalarial agents of the artemisinin-type and FE(III)-heme by electrospray mass spectrometry and collisional activation tandem mass spectrometry.

In this study, we demonstrate, using electrospray ionization mass spectrometry (ESI-MS) and collision-induced dissociation tandem mass spectrometry (ESI-MS/CID/MS), that stable noncovalent complexes can be formed between Fe(III)-heme and antimalarial agents, i.e., quinine, artemisinin, and the artemisinin derivatives, dihydroartemisinin, alpha- and beta-artemether, and beta-arteether. Differences in the binding behavior of the examined drugs with Fe(III)-heme and the stability of the drug-heme complexes are demonstrated. The results show that all tested antimalarial agents form a drug-heme complex with a 1:1 stoichiometry but that quinine also results in a second complex with the heme dimer. ESI-MS performed on mixtures of pairs of various antimalarial agents with heme indicate that quinine binds preferentially to Fe(III)-heme, while ESI-MS/CID/MS shows that the quinine-heme complex is nearly two times more stable than the complexes formed between heme and artemisinin or its derivatives. Moreover, it is found that dihydroartemisinin, the active metabolite of the artemisinin-type drugs in vivo, results in a Na(+)-containing heme-drug complex, which is as stable as the heme-quinine complex. The efficiency of drug-heme binding of artemisinin derivatives is generally lower and the decomposition under CID higher compared with quinine, but these parameters are within the same order of magnitude. These results suggest that the efficiency of antimalarial agents of the artemisinin-type to form noncovalent complexes with Fe(III)-heme is comparable with that of the traditional antimalarial agent, quinine. Our study illustrates that electrospray ionization mass spectrometry and collision-induced dissociation tandem mass spectrometry are suitable tools to probe noncovalent interactions between heme and antimalarial agents. The results obtained provide insights into the underlying molecular modes of action of the traditional antimalarial agent quinine and of the antimalarials of the artemisinin-type which are currently used to treat severe or multidrug-resistant malaria.

The application of liquid chromatography-electrospray ionization mass spectrometry and collision-induced dissociation in the structural characterization of acylated flavonol O-glycosides from the seeds of Carrichtera annua.

The flavonoid fraction from the seeds of Carrichtera annua was studied using high-performance liquid chromatography simultaneously coupled to a photodiode array detector (LC/UV-DAD) and a mass spectrometer equipped with an electrospray source (LC/ESI-MS). Collision-induced dissociation (CID) mass spectral data obtained off-line by nanospray (nano-ESI) analysis provided a wealth of complementary structural information, which was consistent with structures established by NMR or led to the proposal of base structures of the flavonol O-glycosides present in the Carrichtera annua seed extract. The flavonoid fraction was found to contain 12 structurally related flavonol O-glycosides. Eleven flavonoids, of which several were new compounds, were acylated with one or more benzoyl, feruloyl or sinapoyl groups. These acyl groups gave rise to characteristic product ions in the [M + H](+) and [M + Na](+) CID spectra as well as to radicalar acid-related product ions at high-energy collisional activation. In addition to the characterization of the acyl substituents, the mass spectral data allowed the identification of the aglycone, the determination of the base structure and the differentiation of several positional isomers.

Complement-inhibiting iridoids from Morinda morindoides.

Morinda morindoides (Baker) Milne-Redhead (syn. Gaertnera morindoides Bak.) is one of the most popular medicinal plants in the Democratic Republic of Congo. In relation to its traditional use against rheumatic pains, fractionation of both the EtOAc- and the n-BuOH-soluble fraction of the 80% MeOH extract of the leaves, guided by the anticomplementary activity on the classical activation pathway, yielded eight novel iridoids (1-8), all containing a spirolactone functionality. Their structure was elucidated using spectroscopic methods. Gaertneroside 1, acetylgaertneroside 2, and gaertneric acid 5 were found to inhibit the activation of the classical pathway of the complement system, with IC(50) values between 58 and 69 microM. In addition to the biologically active flavonoids reported before from the same plant, these complement-inhibiting iridoids may contribute at least in part to the traditional use against rheumatic pains.

Structural characterization of chromone C-glucosides in a toxic herbal remedy.

Two novel compounds, 8-C-D-glucopyranosyl-7-hydroxy-5-methylchromone-2-carboxylic acid and a 2-O'-p-coumaroyl derivative thereof, were identified in a herbal tea that caused severe vomiting in a South African patient who had taken the traditional remedy to clean his stomach. For structural characterization, electrospray (ES) ionization in combination with collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) were used, as well as UV and nuclear magnetic resonance (NMR) spectroscopy. Specific ions or neutral losses generated under conditions of ES-MS/CID/MS permitted the establishment of structural features such as the free carboxyl group, the C-hexosidic part and the p-coumaroyl group. NMR spectroscopy was necessary to support the structure of the chromone-type aglycone and the glucosidic parts. Since the compounds are structurally related to aloesin and aloeresin A, which are chemotaxonomic markers of Aloe species, and have not been previously reported, we propose that they were formed by oxidative degradation during preparation of the herbal tea from an Aloe species or during its storage.

In vitro inhibition of [3H]-angiotensin II binding on the human AT1 receptor by proanthocyanidins from Guazuma ulmifolia bark.

A bioassay-guided fractionation of the 70% acetone extract of the bark of Guazuma ulmifolia Lam. on the inhibition of angiotensin II binding to the AT 1 receptor led to the isolation and identification of bioactive oligomeric and polymeric proanthocyanidins consisting mainly of (-)-epicatechin units. The displacement of [3H]-angiotensin II binding was dose-dependent and correlated with the degree of polymerization of the different fractions containing proanthocyanidins. A strong displacement was seen for the residual fraction suggesting that the most active substances corresponding to the highly polymerized proanthocyanidins. Angiotensin II AT 1 receptor binding might be considered as a potentially interesting biological activity of proanthocyanidins contributing to the very broad spectrum of biological activities of the condensed tannins.

Inhibitory activity on binding of specific ligands to the human angiotensin II AT(1) and endothelin 1 ET(A) receptors: bioactive benzo[c]phenanthridine alkaloids from the root of Bocconia frutescens.

A bioassay-guided fractionation of the 80 % ethanolic extract from Bocconia frutescens L. roots, showing a dose-dependent inhibitory effect towards both [(3)H]-angiotensin II and [(3)H]-BQ-123 binding to the human angiotensin II AT 1 and endothelin 1 ET(A) receptors, led to an alkaloidal subfraction as the only responsible fraction for the activity of the whole extract. Among the alkaloids present in this fraction sanguinarine and chelerythrine were significant inhibitors of [(3)H]-angiotensin II binding (hAT 1 receptor), with IC(50) values within the micromolar range. On the contrary, the [(3)H]-BQ-123 binding (ET(A) receptor) was only weakly inhibited. Moreover, other members of the isoquinoline alkaloid family such as chelidonine and some protoberberine alkaloids exhibited no affinity for the two receptors. The present work shows the possible structure-activity relationship for these benzophenanthridine alkaloids on a screening bioassay using both stably transfected Chinese hamster ovary (CHO) and the human neuroblastoma SK-N-MC cells. Furthermore, the ability of these compounds to block AT(1) and/or ET(A) receptors may provide some justification for the traditional use of Bocconia frutescens L. to control hypertension.

Synthesis, cytotoxicity, and antiplasmodial and antitrypanosomal activity of new neocryptolepine derivatives.

On the basis of the original lead neocryptolepine or 5-methyl-5H-indolo[2,3-b]quinoline, an alkaloid from Cryptolepis sanguinolenta, derivatives were prepared using a biradical cyclization methodology. Starting from easily accessible educts, this approach allowed the synthesis of hitherto unknown compounds with a varied substitution pattern. As a result of steric hindrance, preferential formation of the 3-substituted isomers over the 1-substituted isomers was observed when cyclizing N-(3-substituted-phenyl)-N'-[2-(2-trimethylsilylethynyl)phenyl]carbodiimides. All compounds were evaluated for their activity against chloroquine-sensitive as well as chloroquine-resistant Plasmodium falciparum strains, for their activity against Trypanosoma brucei and T. cruzi, and for their cytotoxicity on human MRC-5 cells. Mechanisms of action were investigated by testing heme complexation using ESI-MS, inhibition of beta-hematin formation, DNA interactions (DNA-methyl green assay and linear dichroism), and inhibition of human topoisomerase II. Neocryptolepine derivatives with a higher antiplasmodial activity and a lower cytotoxicity than the original lead have been obtained. This selective antiplasmodial activity was associated with inhibition of beta-hematin formation. 2-Bromoneocryptolepine was the most selective compound with an IC(50) value against chloroquine-resistant P. falciparum of 4.0 microM in the absence of cytotoxicity (IC(50) > 32 microM). Although cryptolepine, a known lead for antimalarials also originally isolated from Cryptolepis sanguinolenta, was more active (IC(50) = 2.0 microM), 2-bromoneocryptolepine showed a low affinity for DNA and no inhibition of human topoisomerase II, in contrast to cryptolepine. Although some neocryptolepine derivatives showed a higher antiplasmodial activity than 2-bromocryptolepine, these compounds also showed a higher affinity for DNA and/or a more pronounced cytotoxicity. Therefore, 2-bromoneocryptolepine is considered as the most promising lead from the present work for new antimalarial agents. In addition, 2-bromo-, 2-nitro-, and 2-methoxy-9-cyanoneocryptolepine exhibited antitrypanosomal activity in the micromolar range in the absence of obvious cytotoxicity.

Resonant electron capture mass spectrometry of free fatty acids: examination of ion structures using deuterium-labeled fatty acids and collisional activation.

The structures of [M-H](-) ions generated from free fatty acids in resonant electron capture at energies of 1.2 and 7.2 eV were investigated using deuterium-labeled isotopomers and collision-induced dissociation. The [M-H](- small middle dot) ions occur in both a carboxylate anion and a carbanion form. While the formation of the carboxylate anion at 1.2 eV involves the loss of a carboxylic hydrogen, that at 7.2 eV involves the loss of a hydrogen from different positions in the aliphatic chain followed by a rearrangement of a carboxylic hydrogen on to the radical site in the chain. The [M-H-H(2)O](-) ion which is a minor ion in the resonant electron capture spectrum at 7.2 eV is shown to be a precursor for the charge-remote fragment ions corresponding to formal losses of a hydrogen and elements of alkanes. The [M-H-H(2)O](-) ion corresponding to the second major ion in the resonant electron capture spectrum at 7.2 eV is demonstrated to be consistent with a cyclopentanone anion structure. On the basis of new insights obtained in the present study and taking into account previous results, an updated proposal is presented for the mechanism of charge-remote fragmentation which operates in resonant electron capture of free fatty acids at 7.2 eV.

Herbal remedy-associated acute renal failure secondary to Cape aloes.

Use of traditional herbal remedies is common in Africa, and many patients who visit traditional healers do not need to resort to Western medicine. Acute renal failure is one of the most serious complications resulting from the use of traditional remedies, however, which accounts for 35% of all cases of acute renal failure in Africa. Traditional remedies rarely have been analyzed, and little is known about their nephrotoxicity. We report a case of a 47-year-old man from Soweto, South Africa, who developed acute oliguric renal failure and liver dysfunction after ingestion of an herbal remedy. The patient's renal function recovered slowly, and dialysis was discontinued after several weeks, although serum creatinine did not return to the normal range. Mass spectrometric and chromatographic analysis of the herbal remedy used by the patient revealed the presence of Cape aloes, a previously described nephrotoxin.