Metabolite Profiling of Anti‐Addictive Alkaloids from Four Mexican Tabernaemontana Species and the Entheogenic African Shrub Tabernanthe iboga (Apocynaceae)

Ibogaine and other ibogan type alkaloids present anti‐addictive effects against several drugs of abuse and occur in different species of the Apocynaceae family. In this work, we used gas chromatography‐mass spectrometry (GC/MS) and principal component analysis (PCA) in order to compare the alkaloid profiles of the root and stem barks of four Mexican Tabernaemontana species with the root bark of the entheogenic African shrub Tabernanthe iboga. PCA demonstrated that separation between species could be attributed to quantitative differences of the major alkaloids, coronaridine, ibogamine, voacangine, and ibogaine. While T. iboga mainly presented high concentrations of ibogaine, Tabernaemontana samples either showed a predominance of voacangine and ibogaine, or coronaridine and ibogamine, respectively. The results illustrate the phytochemical proximity between both genera and confirm previous suggestions that Mexican Tabernaemontana species are viable sources of anti‐addictive compounds.     

Quantitative Evaluation of a Mexican and a Ghanaian Tabernaemontana Species as Alternatives to Voacanga africana for the Production of Antiaddictive Ibogan Type Alkaloids

In continuation of our efforts to provide quantitative information on antiaddictive ibogan type alkaloid‐producing Tabernaemontana species, we used gas chromatography‐mass spectrometry (GC/MS) to compare the alkaloid profiles of the barks and/or leaves of one Mexican and one African species – T. arborea and T. crassa, respectively, with the primary sources of commercially available semisynthetic ibogaine, Voacanga africana root and stem bark. The qualitative and quantitative similarities between T. arborea and V. africana barks consolidate previous reports regarding the potential of the former as a promising alternative source of voacangine and ibogaine. The results also suggest that T. crassa could be used to produce conopharyngine and ibogaline, two compounds with the same basic skeletal structure and possibly similar antiaddictive properties as ibogaine.     

Indole alkaloids from Tabernaemontana australis (Muell. Arg) Miers that inhibit acetylcholinesterase enzyme

Ten indole alkaloids from the chloroform extract of stalk of Tabernaemontana australis (Muell. Arg) Miers were tentatively identified by GC-MS, viz., coronaridine (1), voacangine (2), voacangine hydroxyindolenine (3), rupicoline (4), ibogamine (5), ibogaine (6), ibogaline (7), desethyl-voacangine (8), voachalotine (9), and affinisine (10). Of these, the first four were isolated by silica gel open column chromatography, identified by uni- and bidimensional NMR, IR, MS and showed anti-cholinesterasic activity at the same concentration as the reference compounds physostigmine and galanthamine (detection limit of 0.01mM) by TLC assay using the modified Ellman's method.     

Quantification of Anti‐Addictive Alkaloids Ibogaine and Voacangine in In Vivo‐ and In Vitro‐Grown Plants of Two Mexican Tabernaemontana Species

Tabernaemontana alba and Tabernaemontana arborea are Apocynaceae species used in Mexican traditional medicine for which little phytochemical information exists. In this study, preliminary gas chromatography/mass spectrometry analyses of different organs obtained from wild plants of both species identified a total of 10 monoterpenoid indole alkaloids (MIAs) and one simple indole alkaloid, nine of which were reported for the first time in these species. Furthermore, callus cultures were established from T. alba leaf explants and regeneration of whole plants was accomplished via somatic embryogenesis. The anti‐addictive MIAs ibogaine and voacangine were then quantified by gas chromatography with flame ionization detection in wild plants of both species, as well as greenhouse‐grown plants, in vitro‐grown plantlets and embryogenic callus of T. alba. Ibogaine and voacangine were present in most samples taken from the whole plants of both species, with stem and root barks showing the highest concentrations. No alkaloids were detected in callus samples. It was concluded that T. alba and T. arborea are potentially viable sources of ibogaine and voacangine, and that these MIAs can be produced through somatic embryogenesis and whole plant regeneration of T. alba. Approaches to increase MIA yields in whole plants and to achieve alkaloid production directly in cell cultures are discussed.     

New alkaloids from Tabernaemontana divaricata

A re-investigation of the root bark of Tabernaemontana divaricata resulted in the isolation of 18 compounds, viz. α-amyrin acetate, lupeol acetate, α-amyrin, lupeol, cycloartenol, β-sitosterol, campesterol, benzoic acid, aurantiamide acetate, coronaridine, coronaridine hydroxyindolenine (1), ibogamine, 5-hydroxy-6-oxocoronaridine (6), 5-oxocoronaridine (9), 6-oxocoronaridine (10), (±)-19-hydroxycoronaridine (11), 3-oxocoronaridine (12) and voacamine. The alkaloids 6, 9, 10 and 11 are new compounds, whereas cycloartenol, campesterol, ibogamine, benzoic acid, aurantiamide acetate and coronaridine hydroxyindolenine are reported from this plant for the first time.     

Cytotoxicity and genotoxicity of coronaridine from Tabernaemontana catharinensis A.DC in a human laryngeal epithelial carcinoma cell line (Hep-2)

Cancer has become a major public health problem worldwide and the number of deaths due to this disease is increasing almost exponentially. In the constant search for new treatments, natural products of plant origin have provided a variety of new compounds to be explored as antitumor agents. Tabernaemontana catharinensis is a medicinal plant that produces alkaloids with expressive antitumor activity, such as heyneanine, coronaridine and voacangine. The aim of present study was firstly to screen the cytotoxic activity of the indole alkaloids heyneanine, coronaridine and voacangine against HeLa (human cervix tumor), 3T3 (normal mouse embryo fibroblasts), Hep-2 (human laryngeal epithelial carcinoma) and B-16 (murine skin) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); and secondly to analyze the apoptotic activity, cell membrane damage and genotoxicity of the compound that showed the best cytotoxic activity against the tumor cell lines tested. Coronaridine was the one that exhibited greater cytotoxic activity in the laryngeal carcinoma cell line Hep-2 (IC₅₀ = 54.47 μg/mL) than the other alkaloids tested (voacangine IC₅₀ = 159.33 g/mL, and heyneanine IC₅₀ = 689.45 μg/mL). Coronaridine induced apoptosis in cell lines 3T3 and Hep-2, even at high concentrations. The evaluation of genotoxicity by comet assay showed further that coronaridine caused minimal DNA damage in the Hep-2 tumor cell line, and the LDH test showed that it did not affect the plasma membrane. These results suggest that further investigation of coronaridine as an antitumor agent has merit.     

Alkaloids of stem and rootbark of Tabernaemontana dichotoma

During chemical investigation for compounds possessing biological activity in the stem and rootbark of Tabernaemontana dichotoma, 22 alkaloids were isolated. Ten monomeric alkaloids were identified, viz. (?)-apparicine, coronaridine, 3-oxocoronaridine, 3-ketopropylcoronaridine, 19R-heyneanine, 3-ketopropyl-19R-heyneanine, ibogamine, isomethuenine, perivine and vobasine. Two of the monomeric alkaloids isolated were new, one was identified as 3,19R-oxidocoronaridine and the other one is not yet identified. The other ten alkaloids isolated were dimeric compounds, three were identified as tabernamine, voacamine and 3′R/S-hydroxyvoacamine. Five of the dimers were new alkaloids related to tabernamine and ervahanine type structures and identified as 3′R/S-hydroxytabernamine, 3′R/S-hydroxy-N₄-demethyltabernamine, N₄-demethyltabernamine and 3′R/S-hydroxy-N₄-demethytervahanine A and B. The remaining two alkaloids are partially characterized.     

Bio-guided search of active indole alkaloids from Tabernaemontana catharinensis: Antitumour activity,toxicity in silico and molecular modelling studies

Active plant metabolites have been used as prototype drugs. In this context, Tabernaemontana catharinensis (Apocynaceae) has been highlighted because of the presence of active indole alkaloids. Thus, this study aims the bio-guided search of T. catharinensis cytotoxic alkaloids. The chemical composition was identified by high-resolution mass spectrometry, and fractionation was performed by open column and preparative thin-layer chromatography, from plant stems. The enriched fractions were tested in vitro in tumour cells A375 (melanoma cell line) and A549 (adenocarcinomic human alveolar basal epithelial cells), and non-tumour Vero cells (African green monkey kidney epithelial cells). The alkaloids identified as active were submitted to in silico toxicity prediction by ADME-Tox and OSIRIS programs and, also, to molecular docking, using topoisomerase I (PDB ID: 1SC7) by iGEMDOCK. As a result, six sub-fractions were obtained, which were identified as containing 16-epi-affinine, 12-methoxy-n-methyl-voachalotine, affinisine, voachalotine, coronaridine hydroxyindoline and ibogamine, respectively. The affinisine-containing sub-fraction showed selective toxicity against A375, with an IC₅₀ of 11.73 µg mL⁻¹, and no cytotoxicity against normal cells (Vero). From the in silico toxicity test results, all indole alkaloid compounds had a low toxicity risk. The molecular docking data provided structural models and binding affinities of the plant’s indole alkaloids and topoisomerase I. In summary, this bio-guided search revealed that the indole alkaloids from T. catharinensis display selective cytotoxicity in A375 tumour cells and toxicity in silico. Particularly, affinisine might be a chemotherapeutic for A375 melanoma cells.     

Effects of ibogaine,and cocaine and morphine after ibogaine,on ventral tegmental dopamine neurons

Ibogaine, an indole containing alkaloid, has been shown to reduce the rate of injection of morphine and cocaine in self-administration protocols. Since morphine- and cocaine-induced modulation of dopamine release is impulse dependent and essential for their reinforcing effects, disruption of dopamine neuronal activity by ibogaine could explain its purported ‘antiaddictive’ properties. Therefore, the present study was designed to determine: (1) the acute effects of ibogaine on the activity of VTA dopamine neurons, and (2) whether ibogaine pretreatment causes a persistent modification of the dopamine neuronal response to morphine and cocaine. Extracellular recordings in anesthetized animals found that intravenous ibogaine markedly excited VTA dopamine neuronal firing. However, ibogaine pretreatment (6–8 hr and 19 hr before) failed to alter either the spontaneous activity of VTA neurons, or the response of these dopamine neurons to morphine or cocaine. Thus, ibogaine's excitatory effect on VTA neurons is not longlasting nor does it persistently alter cocaine- or morphine-induced changes in dopamine neuron impulse activity. Therefore, other mechanisms must be explored to account for the proposed antiaddictive properties of ibogaine.     

Long-Lasting Ibogaine Protection against NMDA-Induced Convulsions in Mice

Ibogaine, a putative antiaddictive drug, is remarkable in its apparent ability to downgrade withdrawal symptoms and drug craving for extended periods of time after a single dose. Ibogaine acts as a non-competitive NMDA receptor antagonist, while NMDA has been implicated in long lasting changes in neuronal function and in the physiological basis of drug addiction. The purpose of this study was to verify if persistent changes in NMDA receptors could be shown in vivo and in vitro after a single administration of ibogaine. The time course of ibogaine effects were examined on NMDA-induced seizures and [³H] MK-801 binding to cortical membranes in mice 30min, 24, 48, and 72h post treatment. Ibogaine (80 mg/kg, ip) was effective in inhibiting convulsions induced by NMDA at 24 and 72 hours post administration. Likewise, [³H] MK-801 binding was significantly decreased at 24 and 72 h post ibogaine. No significant differences from controls were found at 30min or 48h post ibogaine. This long lasting and complex pattern of modulation of NMDA receptors prompted by a single dose of ibogaine may be associated to its antiaddictive properties.     

Induction of triterpene biosynthesis by elicitors in suspension cultures of Tabernaemontana species

Treatment of suspension cultures of some Tabernaemontana species (Apocynaceae) with elicitors (e.g. cellulase, Candida albicans) result in a rapid de novo production of antimicrobial active triterpenes. The triterpenes are identified as ursene carboxylic acid derivatives. These triterpenes are not produced by an elicited cell suspension culture of Catharanthus roseus, another Apocynaceae.     

Ochtodes searlesii sp. nov. (Gigartinales,Rhodophyta), from the Pacific tropical coast of Mexico,based on morphological and molecular evidence

Ochtodes searlesii Mendoza‐González, Mateo‐Cid et Sentíes sp. nov. is described from Michoacán, tropical Mexican Pacific, on the basis of comparative morphology and rbcL sequence analysis. It is distinguished from other Ochtodes species by its erect axes arising from an encrusting base, its small terete fronds, regularly dichotomously branched axes, and obliquely divided zonate tetrasporangia. Phylogenetic analyses showed that three Pacific Mexican samples, from Caletilla, Zapote and La Majahuita (Michoacán), were identical and formed a distinctive and well supported Clade segregated from other species of Ochtodes from Brazil, Cuba, Ecuador, Guadeloupe and Mexico. The Mexican entity is morphologically distinct from other Ochtodes species as well. On this basis we propose a new Ochtodes species.     

Alcaloïdes des feuilles de Voacanga thouarsii

Leaves of Voacanga thouarsii Roem. et Schult. yielded 3 indole alkaloids (ibogaine, voacangine, voacristine) and 12 ‘bis-indole’ alkaloids. Three of the latter are known (vobtusine, vobtusinelactone and subsessiline) and nine are new.     

Inhibition of platelet aggregation and thrombosis by indole alkaloids isolated from the edible insect Protaetia brevitarsis seulensis (Kolbe)

Protaetia brevitarsis seulensis (Kolbe) has been temporarily registered as a food material by the Ministry of Food and Drug Safety of Korea (MFDS). The current study aimed to discover small antithrombotic molecules from this edible insect. Five indole alkaloids, 5‐hydroxyindolin‐2‐one ( 1 ), (1R,3S)‐1‐methyl‐1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid ( 2 ), (1S,3S)‐1‐methyl‐1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid ( 3 ), (3S)‐1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid ( 4 ) and L‐tryptophan ( 5 ), were isolated from the insect. Among them, compounds 1 and 2 prolonged aPTT and PT and impaired thrombin and FXa generation on HUVEC surface. Moreover, these compounds inhibited platelet aggregation. Antithrombotic effects of compounds 1 and 2 were further confirmed in pre‐clinical models of pulmonary embolism and arterial thrombosis. Collectively, these results demonstrated that compounds 1 and 2 could be effective antithrombotic agents and serve as new scaffolds for the development of antithrombotic drug.     

Tree fruiting phenology in Kalinzu Forest, Uganda

The spatial and temporal variations in species composition and abundance of trees that fruited in mechanically logged, intensively pit‐sawn and essentially undisturbed forest areas in Kalinzu Forest (0°17′S, 0°30′S and 30°00′, 30°07′E) were assessed. The duration and frequency of fruiting episodes of selected pioneer, understorey and canopy tree species were also determined. These phenology aspects were monitored for 18 months (between February 1997 and July 1998) in 99 plots, each 20 m × 20 m in size. Eighty species consisting of 1489 trees fruited during the study. Most of the species were recorded in the undisturbed forest area and least in the mechanically logged areas. Monthly number of individuals and species that fruited in each forest condition were significantly related to monthly rainfall. Two pioneer species (Musanga leo‐errerae and Trema orientalis), one sub‐canopy (Funtumia africana) and canopy (Parinari excelsa) species had extended fruiting phases (>5 months). Two understorey species (Oxyancius speciosus and Tabernaemontana spp.) had an extended phase while the third (Teclea nobilis) had a short (≤5 months) fruiting phase. Of the selected species, only Strombosia scheffleri (a sub‐canopy species) and P. excelsa had more than one fruiting episode.     

Phylogenetic relationships of Mexican minnows of the genus Notropis (Actinopterygii, Cyprinidae)

We conducted phylogenetic analyses based on complete mitochondrial cytochrome b gene sequences among southern and central Mexican cyprinid species, included in the genera Notropis and Hybopsis. In addition 15 northern species of the genera Notropis and Hybopsis were included in the analyses in order to place the Mexican species into a larger phylogenetic framework. The phylogenetic relationships supported the existence of five major clades: (1) including species of the subgenus Alburnops of the genus Notropis plus N. shumardi; (2) species of the subgenus Notropis; (3) species of the genus Hybopsis; (4) species of the N. texanus + N. volucellus species group of the genus Notropis; (5) Mexican endemic species of the genus Notropis plus the genus Yuriria. Previous phylogenetic inferences based on morphological characters resolved the Mexican minnows analysed as N. sallaei, N. calientis, N. boucardi and Y. alta, non‐monophyletic. According to our cytochrome b evidence all Mexican minnows of the genera Notropis and Yuriria formed a monophyletic group with respect to the northern species of the genera Notropis and Hybopsis. Within the Mexican clade, three well‐supported clades were identified: the first included the closely related species N. moralesi and N. boucardi, which occur in three independent drainages of south Mexico; the second consisted of two different lineages, N. imeldae and an undescribed species of Notropis, inhabiting two independent drainages of south Mexico; the third comprised two central Mexican Notropis species (N. calientis and N. sallaei) and the Y. alta populations. Based on this study and pending a more extensive taxonomic revision of the genus Notropis, we adopt the conservative criterion of considering all Notropis species from southern and central Mexico examined, including Y. alta, as belonging to the genus Notropis. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80 , 323–337.     

Diversity and distribution of Laurencia sensu stricto (Rhodomelaceae,Rhodophyta) from the Mexican Pacific,including L. mutueae sp. nov.

The alpha diversity of Laurencia sensu stricto, as well as the majority of the other algal groups along the Mexican Pacific, was underestimated because very few studies had incorporated the use of molecular markers in taxonomic studies, which would allow a more realistic estimation of diversity, incluing new records and new species. The records of Laurencia s.s. for the Eastern Pacific (California, USA to Chile) currently represent 23% of its richness worldwide. In this work, we proposed an updated census of the currently recognized species of Laurencia s.s. for the Mexican Pacific. This checklist reported the occurrence of 22 species that represent 16% of the 137 species worldly recorded. We further included taxonomic remarks, type localities, and a detailed distribution for each species. In addition, we proposed a new species for this region, Laurencia mutueae sp. nov., based on morphological observation as well as phylogenetic analyses. The distribution in the Mexican Pacific revealed that the highest records of species were for the sub‐temperate region, while the localities along the Mexican tropical Pacific were poorly recorded. The scarcity of floristic affinities between the eastern and the western and central parts of the North Pacific Ocean was hypothesized to be a consequence of a strong isolation and hard barriers, such as the water masses between these regions.     

Indole alkaloids from cell suspension cultures of Tabernaemontana divaricata and Tabernanthe iboga

From cell suspension cultures of Tabernaemontana divaricata and Tabernanthe iboga grown under standard conditions, six monoterpenoid indole alkaloids have been isolated and identified. T. divaricata synthesized apparicine, catharanthine, coronaridine, conoflorine, tubotaiwine and vinervine, whereas T. iboga produced tubotaiwine and conoflorine. Both cultures are a reasonable source for conoflorine, which is expected to be a good candidate for studying the mechanism of Aspidosperma type alkaloid formation at the cell-free level.