LC-MS/MS quantitative determination of Tetrapterys mucronata alkaloids, a plant occasionally used in ayahuasca preparation.

INTRODUCTION: Tetrapterys mucronata Cav. (Malpighiaceae) is a plant used in some regions of Brazil in the preparation of ayahuasca. OBJECTIVE: To determine the content of the main tryptamine alkaloids in the stem bark of T. mucronata Cav. and assess their possible toxic and hallucinogenic properties based on the doses found in a water decoction that mimics the ayahuasca preparation. METHODS: Four alkaloids previously described for their toxic and hallucinogenic properties were quantitated by multiple reaction monitoring HPLC combined with electrospray ionisation and tandem MS (HPLC-ESI/MS/MS) in the water decoction and ethanolic extracts from the bark of T. mucronata. RESULTS: Exhaustive extraction of the stem barks with ethanol revealed the following alkaloid levels: bufotenine (1) 3.26 ± 0.31 mg/g, 5-methoxy-N-methyltryptamine (2) 0.88 ± 0.08 mg/g, 5-methoxy-bufotenine (3) 3.07 ± 0.22 mg/g and 2-methyl-6-methoxy-1,2,3,4-tetrahydro-ß-carboline (4) 0.14 ± 0.004 mg/g. The water decoction presented slightly lower levels, ranging between 2.32 ± 0.14, 0.50 ± 0.04, 1.53 ± 0.09 and 0.10 ± 0.01 mg/g for (1), (2), (3) and (4) respectively. CONCLUSIONS: The HPLC-ESI/MS/MS quantitation revealed significant alkaloid levels, in particular for bufotenine and 5-methoxy-bufotenine. As such compounds are known for their toxic and hallucinogenic properties, these results indicate that the consumption of this plant as an ingredient in ayahuasca preparations may present a risk to consumers.

Anti-inflammatory, antimicrobial and antioxidant activities of Diospyros bipindensis (Gürke) extracts and its main constituents.

ETHNOPHARMACOLOGICAL RELEVANCE: Diospyros bipindensis (Gürke) stem bark is used in Cameroon by Baka Pygmies for the treatment of respiratory disorders. AIM OF THE STUDY: To assess the anti-inflammatory, antibacterial and antioxidant properties of constituents from the bark extracts through bioassay-guided fractionation. MATERIALS AND METHODS: The anti-inflammatory activity of extracts, fractions and pure compounds was assessed through the inhibition of the pro-inflammatory mediator nuclear factor-kappa B (NF-κB) transcriptional activity and nitric oxide (NO) production. DPPH, ABTS and ORAC assays were used for determining the antioxidant properties. The activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Klebsiella pneumoniae, was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by the macrodilution method. RESULTS: The water extract showed antimicrobial activity against S. pneumoniae (MIC: 300 µg/ml) and S. pyogenes (MIC: 300 µg/ml). The dichloromethane extract efficiently inhibited NF-κB transcriptional activity and NO production and exhibited significant antioxidant activity in the ORAC assay. An interesting activity was also found against S. pneumoniae (MIC: 200 µg/ml), S. aureus (MIC: 400 µg/ml) and S. pyogenes (MIC: 200 µg/ml). The phytochemical investigation of the dichloromethane extract afforded plumbagin, canaliculatin, ismailin, betulinic acid and 4-hydroxy-5-methyl-coumarin as the main constituents. Plumbagin and ismailin were found to be responsible for the main biological activities observed. CONCLUSIONS: These results may provide a rational support for the traditional use of Diospyros bipindensis stem bark in the treatment of respiratory disorders, since the anti-inflammatory, antimicrobial and antioxidant compounds isolated from the dichloromethane extract were also present in the traditional water extract.

3-Farnesyl-2-hydroxybenzoic acid is a new anti-Helicobacter pylori compound from Piper multiplinervium.

A new prenylated salicylic acid derivative, 3-farnesyl-2-hydroxy benzoic acid (1), was isolated from the leaves of Piper multiplinervium C. DC. (Piperaceae). It showed anti-Helicobacter pylori activity (MIC 37.5 microg/ml) and antimicrobial activity at MICs between 2.5 and 5 microg/ml against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Mycobacterium smegmatis, Pseudomonas aeruginosa and Candida albicans. Its structure was elucidated by means of MS, 1H and 13C NMR. The ethnomedical claim of Piper multiplinervium to treat stomach aches by the Kuna Indians of Panama may be justified by anti-Helicobacter pylori activity of its MeOH extract.

On-line identification of unstable iridoids from Jamesbrittenia fodina by HPLC-MS and HPLC-NMR.

HPLC-UV-MS analysis of the methanol extract of Jamesbrittenia fodina (Wild) O. M. Hilliard (Scrophulariaceae) revealed the presence of different iridoid cinnamic esters; however, isolation of these constituents was prevented by instability problems. HPLC-UV-MS and HPLC-NMR analysis of the mixtures obtained after a tentative isolation indicated that, in the first instance, instability was due to a light-induced cis/trans isomerisation of the cinnamic moieties. Further investigation of related compounds showed an additional instability problem linked to other chemical transformations. A detailed HPLC-NMR-MS study of these fractions demonstrated that the modifications occurred on the rhamnose moiety of these iridoids. It could be concluded that the second type of instability was attributable to transesterification of the cinnamic moiety on the rhamnose unit. The recording of stop-flow HPLC-NMR spectra for specific HPLC peaks permitted the direct monitoring of these transformations. Based on these on-line data, six new unstable aucubin derivatives were efficiently characterised.

On-line identification of the bioactive compounds from Blumea gariepina by HPLC-UV-MS and HPLC-UV-NMR, combined with HPLC-micro-fractionation.

The dichloromethane extract of the aerial parts of Blumea gariepina (Asteraceae) was shown to be active against the phytopathogenic fungus Cladosporium cucumerinum and to inhibit acetylcholinesterase. In order rapidly to identify the active principles, the crude extract was analysed by on-flow HPLC-1H-NMR. HPLC-micro-fractionation was performed and all peaks collected were submitted to assays against C. cucumerinum and acetylcholinesterase. By this means, the biological activities could be efficiently associated with selected HPLC peaks. Complementary on-line structural data for all peaks of interest in the crude extract were obtained from HPLC-MS and from HPLC-UV with post-column addition of UV shift reagents. This chemical screening strategy with integrated bioassays permitted the on-line identification of a number of constituents and gave useful information for an efficient isolation procedure.

On-line identification of tropane alkaloids from Erythroxylum vacciniifolium by liquid chromatography-UV detection-multiple mass spectrometry and liquid chromatography-nuclear magnetic resonance spectrometry.

The bark of catuaba (Erythroxylum vacciniifolium Martius, Erythroxylaceae), a tree native to the northern part of Brazil, was investigated for its alkaloid content. With the aim of obtaining preliminary structure information on-line, the alkaloid extract was analysed by high-performance liquid chromatography coupled to diode array UV detection, to mass spectrometry and to nuclear magnetic resonance. Interpretation of on-line spectroscopic data obtained from this extract led to structural elucidation of six new alkaloids and partial identification of 18 potentially original alkaloids bearing the same tropane skeleton esterified in positions 3 and 6 by 1-methyl-1H-pyrrol-2-carboxylic acid and/or 4-hydroxy-3,5-dimethoxybenzoic acid.

Heliotropamide, a novel oxopyrrolidine-3-carboxamide from Heliotropium ovalifolium.

Heliotropamide (1), a new alkaloid with a novel oxopyrrolidine-3-carboxamide central moiety, has been isolated as the major product of the dicholoromethane extract of Heliotropium ovalifolium aerial parts. Its structure was elucidated by spectrometric methods including ESI-HR, EI, D/CI mass spectrometry, (1)H, (13)C, and 2D NMR experiments, and chemical derivatization. Neither heliotropamide nor its acetylated derivative (1a) showed any antifungal activity against Cladosporium cucumerinum and Candida albicans, antibacterial activity against Bacillus subtilis, radical-scavenging properties in the DPPH test, or inhibitory potential toward acetylcholinesterase.

On-line identification of unstable catalpol derivatives from Jamesbrittenia fodina by LC-mS and LC-NMR.

LC-UV-MS analysis of the methanol extract of Jamesbrittenia fodina O. M. Hilliard (Scrophulariaceae) revealed the presence of cinnamic ester derivatives. Two isomeric pairs of these constituents were detected, but could not be isolated. In order to identify these unstable compounds, LC-1H-NMR spectra were obtained for each individual isomer and standard NMR measurements were performed in-mixture. The spectra clearly demonstrated that the fractions consisted of mixtures of cis and trans cinnamoyl catalpol glycoside esters.

Determination of the absolute configuration of 6-alkylated alpha-pyrones from Ravensara crassifolia by LC-NMR.

The absolute configuration of asymmetric centres of two alpha-pyrones isolated from Ravensara crassifolia was determined using the Mosher method. The conventional analysis of the purified ester derivatives by 1H-NMR was replaced by a rapid and sensitive method in which the alpha-pyrones were analysed under isocratic reversed-phase LC-NMR conditions prior to and after derivatisation reactions. Comparison of the LC-1H-NMR spectra of the actual alpha-pyrones with those of the corresponding Mosher's esters recorded in the acetonitrile:deuterated water solvent system exhibited shifts comparable with those obtained using conventional deuterated solvents. Based on the shifts recorded, determination of the absolute configuration was possible by application of Mosher rules. The use of LC-NMR has permitted a direct analysis of crude reaction mixtures containing less than 50 microg of the starting material. Completion of the reaction was checked by LC-MS and the crude reaction mixture was analysed by stop-flow LC-NMR. This methodology seems very promising for the determination at the micro-scale level of the absolute configuration of natural products which are available only in very small amounts.

On-line identification of the antifungal constituents of Erythrina vogelii by liquid chromatography with tandem mass spectrometry, ultraviolet absorbance detection and nuclear magnetic resonance spectrometry combined with liquid chromatographic micro-fractionation.

In our continuing search for new antifungal agents of plant origin, the investigation of Erythrina vogelii Hook. f. (Leguminosae), a plant used in the traditional medicine of Ivory Coast to treat various infectious ailments, was undertaken. In order to rapidly identify the active principles, the crude extract was analysed by low-flow LC-1H nuclear magnetic resonance spectrometry (NMR) which gave a sensitive detection of all the main peaks. LC microfractionation was performed just after LC-NMR detection and all peaks collected were submitted to antifungal bioautography assays against Cladosporium cucumerinum. By this means, the antifungal activity could be efficiently linked to three of the LC peaks. In order to obtain complementary on-line structural information for all peaks of interest, high-resolution LC-MS-MS together with LC-UV with post-column addition of UV shifts reagents was undertaken on the crude extract. This chemical screening strategy with integrated antifungal bioassays has permitted the on-line identification of numerous constituents and has given useful information for an efficient peak-guided isolation procedure.

Three new prenylated isoflavonoids from the root bark of Erythrina vogelii.

Phytochemical investigation of the CH2Cl2 extract of Erythrina vogelii led to the isolation of five isoflavonoids. Three prenylated isoflavonoids are new natural compounds. The isolation of the antifungal compounds was monitored by inhibition of the growth of Cladosporium cucumerinum in a direct TLC bioautographic assay. The structures of these compounds were elucidated by spectroscopic techniques.

Quinones from Heliotropium ovalifolium.

Two new benzoquinones, heliotropinones A and B, have been isolated from the aerial parts of Heliotropium ovalifolium. Their structures were elucidated by spectrometric methods including high resolution electrospray ionization (ESI-HR), EI mass spectrometry, 1H, 13C and 2D NMR experiments. The two quinones demonstrated antifungal activities against Cladosporium cucumerinum and Candida albicans as well as antibacterial activity against Bacillus subtilis.

Synthesis, spectroscopy, and cytotoxicity of glycosylated acetogenin derivatives as promising molecules for cancer therapy.

Several glycosyl derivatives of squamocin (1) have been synthesized by glycosylation under Lewis acid catalysis with two different 1-O-acetyl sugars. Separation of these compounds has been achieved by HPLC and centrifugal partition chromatography (CPC). A detailed NMR, ESIMS, and LSIMS study allowed complete structural elucidations. The cytotoxic activity of the glycosyl derivatives was investigated and compared with that of squamocin and dihydrosquamocin against human epidermoid carcinoma cells (KB), African green monkey (Cercopithecus aethiops) kidney epithelial cells (VERO), and mouse lymphocytic leukemia cells (L1210). The antiproliferative effects of some derivatives were studied on cell cycles in mouse lymphocytic leukemia cells (L1210).

A new bistetrahydrofuran acetogenin from the roots of Annona salzmanii.

A new bistetrahydrofuran acetogenin, salzmanin (1), was isolated from the MeOH extract of Annona salzmanii, in addition to the known compounds, squamocin, almunequin, bullatalicin, and annonacin. The structure of 1 was elucidated by spectroscopic methods, including LSIMS-MS technique, and confirmed by a chemical transformation. The cytotoxic activity of 1 and squamocin was investigated.

Three new bistetrahydrofuran acetogenins from the seeds of Annona spinescens.

Three new bistetrahydrofuran acetogenins, carolins A-C (1-3), were isolated from the MeOH extract of Annona spinescens in addition to the known compound, squamocin (4). The structures of 1, 2, and 3 were elucidated by spectroscopic methods including LSIMS/MS technique and confirmed by a chemical transformation, The cytotoxic activity of the new compounds 1-3 is reported and discussed in comparison with 4 and the previously isolated spinencin (5).

Pessoine and spinosine, two catecholic berbines from Annona spinescens.

The trunk bark and roots of Annona spinescens have been investigated for their alkaloid content. Two new berbine alkaloids, pessoine (1) and spinosine (2), have been isolated from the bark, and their structures were elucidated by spectroscopic methods. Eight known isoquinoline alkaloids were also obtained. The trypanocidal and antileishmanial activities of these isolated compounds have been investigated.

[Effects of anovulatory steroids on serum levels of zinc and copper]. / Efeitos de esteroides anovulatórios sobre níveis séricos de zinco e cobre.

Mean serum zinc (1.14 micrograms/ml) and copper (2.35 micrograms/ml) in 24 women taking oral contraceptive agents were compared against mean serum zinc (1.25 micrograms/ml) and copper (1.98 micrograms/ml) of 20 women of the same age range who served as controls. Serum zinc was significantly lower while serum copper was significantly higher for women taking the oral contraceptive agents. There was no significant correlation between the serum zinc and copper levels either in the control group (r = 0.04) or in the group taking the contraceptives (r = 0.36).

PIP: Mean serum zinc (1.14 mcg/ml) and copper (2.35 mcg/ml) in 24 women taking oral contraceptives (OCs) were compared against mean serum zinc (1.29 mcg/ml) and copper (1.98 mcg/ml) of 20 same-aged women who served as controls. Serum zinc was significantly lower while serum copper was significantly higher for women taking the OCs. There was no significant correlation between the serum zinc and copper levels either in the control group (r=0.04) or in the group taking the contraceptive (r=0.36). (author's modified)

Efeitos de esteroides anovulatorios sobre niveis sericos de zinco e cobre. / Effects of anovulatory steroids on serum levels of zinc and copper

Niveis sericos medios de zinco (1.14 micrograma/ml) e cobre 2.35 micrograma/ml), em 24 mulheres em uso de anovulatorios orais, foram comparados com niveis sericos medios de zinco (1.29 micrograma/ml) e cobre (1.98 micrograma/ml) de 20 mulheres da mesma faixa etaria que serviram como controles. Os valores encontrados no grupo em uso de anovulatorios foram significantemente mais baixos para o zinco e significantemente mais elevados para o cobre, quando comparados com os valores observados no grupo controle. Nao houve correlacao significativa entre zinco e cobre sericos no grupo controle (r = 0.04) e tampouco no grupo em uso de anovulatorios (r = 0.36)