Reverse pharmacology for developing an anti-malarial phytomedicine. The example of Argemone mexicana.

Classical pharmacology has been the basis for the discovery of new chemical entities with therapeutic effects for decades. In natural product research, compounds are generally tested in vivo only after full in vitro characterization. However drug screening using this methodology is expensive, time-consuming and very often inefficient. Reverse pharmacology, also called bedside-to-bench, is a research approach based on the traditional knowledge and relates to reversing the classical laboratory to clinic pathway to a clinic to laboratory practice. It is a trans-disciplinary approach focused on traditional knowledge, experimental observations and clinical experiences. This paper is an overview of the reverse pharmacology approach applied to the decoction of Argemone mexicana, used as an antimalarial traditional medicine in Mali. A. mexicana appeared as the most effective traditional medicine for the treatment of uncomplicated falciparum malaria in Mali, and the clinical efficacy of the decoction was comparable to artesunate-amodiaquine as previously published. Four stages of the reverse pharmacology process will be described here with a special emphasis on the results for stage 4. Briefly, allocryptopine, protopine and berberine were isolated through bioguided fractionation, and had their identity confirmed by spectroscopic analysis. The three alkaloids showed antiparasitic activity in vitro, of which allocryptopine and protopine were selective towards Plasmodium falciparum. Furthermore, the amount of the three active alkaloids in the decoction was determined by quantitative NMR, and preliminary in vivo assays were conducted. On the basis of these results, the reverse pharmacology approach is discussed and further pharmacokinetic studies appear to be necessary in order to determine whether these alkaloids can be considered as phytochemical markers for quality control and standardization of an improved traditional medicine made with this plant.

Indole alkaloids of Psychotria as multifunctional cholinesterases and monoamine oxidases inhibitors.

Thirteen Psychotria alkaloids were evaluated regarding their interactions with acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and monoamine oxidases A and B (MAO-A and MAO-B), which are enzymatic targets related with neurodegenerative diseases. Two quaternary ß-carboline alkaloids, prunifoleine and 14-oxoprunifoleine, inhibited AChE, BChE and MAO-A with IC(50) values corresponding to 10 and 3.39 µM for AChE, 100 and 11 µM for BChE, and 7.41 and 6.92 µM for MAO-A, respectively. Both compounds seem to behave as noncompetitive AChE inhibitors and time-dependent MAO-A inhibitors. In addition, the monoterpene indole alkaloids (MIAs) angustine, vallesiachotamine lactone, E-vallesiachotamine and Z-vallesiachotamine inhibited BChE and MAO-A with IC(50) values ranging from 3.47 to 14 µM for BChE inhibition and from 0.85 to 2.14 µM for MAO-A inhibition. Among the tested MIAs, angustine is able to inhibit MAO-A in a reversible and competitive way while the three vallesiachotamine-like alkaloids display a time-dependent inhibition on this target. Docking calculations were performed in order to understand the binding mode between the most active ligands and the selected targets. Taken together, our findings established molecular details of AChE, BChE and MAO-A inhibition by quaternary ß-carboline alkaloids and MIAs from Psychotria, suggesting these secondary metabolites are scaffolds for the development of multifunctional compounds against neurodegeneration.

Monoamine oxidase inhibition by monoterpene indole alkaloids and fractions obtained from Psychotria suterella and Psychotria laciniata.

Alkaloid fractions of Psychotria suterella (SAE) and Psychotria laciniata (LAE) as well as two monoterpene indole alkaloids (MIAs) isolated from these fractions were evaluated against monoamine oxidases (MAO-A and -B) obtained from rat brain mitochondria. SAE and LAE were analysed by HPLC-PDA and UHPLC/HR-TOF-MS leading to the identification of the compounds 1, 2, 3 and 4, whose identity was confirmed by NMR analyses. Furthermore, SAE and LAE were submitted to the enzymatic assays, showing a strong activity against MAO-A, characterized by IC(50) values of 1.37 ± 1.05 and 2.02 ± 1.08 µg/mL, respectively. Both extracts were also able to inhibit MAO-B, but in higher concentrations. In a next step, SAE and LAE were fractionated by RP-MPLC affording three and four major fractions, respectively. The RP-MPLC fractions were subsequently tested against MAO-A and -B. The RP-MPLC fractions SAE-F3 and LAE-F4 displayed a strong inhibition against MAO-A with IC(50) values of 0.57 ± 1.12 and 1.05 ± 1.15 µg/mL, respectively. The MIAs 1 and 2 also inhibited MAO-A (IC(50) of 50.04 ± 1.09 and 132.5 ± 1.33 µg/mL, respectively) and -B (IC(50) of 306.6 ± 1.40 and 162.8 ± 1.26 µg/mL, respectively), but in higher concentrations when compared with the fractions. This is the first work describing the effects of MIAs found in neotropical species of Psychotria on MAO activity. The results suggest that species belonging to this genus could consist of an interesting source in the search for new MAO inhibitors.

Isolation and on-line identification of antioxidant compounds from three Baccharis species by HPLC-UV-MS/MS with post-column derivatisation.

The aqueous extracts of aerial parts of Baccharis trimera (Less.) DC., B. crispa Spreng. and B. usterii Heering (Asteraceae) displayed significant radical scavenging activity in a diphenylpicrylhydrazole (DPPH)/TLC assay. In order to rapidly identify the active principles, the crude extracts were analysed by HPLC-UV, and an HPLC-micro-fractionation of the extract of B. usterii was performed. Six quinic acids derivatives (1-6) were isolated from B. usterii by MPLC. The fractions were monitored by DPPH/TLC assay and a series of radical-scavenging quinic acid derivatives could be identified. The comparison of the HPLC profiles of the extracts of B. usterii, B. trimera and B. crispa was performed. In order to obtain complementary on-line structural information for all peaks of interest, HPLC-MS/MS together with HPLC-UV involving post-column addition of UV shift reagents was undertaken on the crude extract. The interpretation of these data permitted the on-line identification of known compounds, some of which are reported for the first time in this plant.

Essential oils from Calyptranthes concinna, C. lucida and C. rubella (Myrtaceae)

Essential oils from Calyptranthes concinna, C. lucida and C. rubella, collected in Southern Brazil, were analyzed by GC and GC/MS. Sixty-two compounds were identified representing about 98 por cento of the oil contents. All samples were rich in cyclic sesquiterpenes (more then 90 por cento), mainly those from cadinane, bisabolane and germacrane cyclization pathway. The mainly components characterized were byclogermacrene (22.1 por cento in C. concinna; 11.7 por cento in C. rubella), cis-calamenene (10.3 por cento in C.concinna), beta-caryophyllene (16.5 por cento in C. rubella; 9.4 por cento in C. lucida), beta-bisabolene (25.5 por cento in C. lucida), spathulenol (15.4 por cento in C. rubella) and caryophyllene oxide (7.6 por cento in C. concinna)