Evolution and diversification of carboxylesterase-like [4+2] cyclases in aspidosperma and iboga alkaloid biosynthesis.

Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent [4+2] cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as "underground metabolites" via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.

Silver nanoparticles from Tabernaemontana divaricate leaf extract: mechanism of action and bio-application for photo degradation of 4-aminopyridine.

Silver nanoparticles (Ag NPs) were synthesised by the reduction of Ag+ to Ag0 in the presence of enol form of flavonoids present in plant extract of Tabernaemontana divaricate (T. divaricate). Prepared Ag NPs were characterised using UV-Vis, XRD, HR-TEM with EDX and XPS techniques. XPS spectra exhibited peaks at 366 eV and 373 eV, which specified spin orbits for Ag 3d3/2, and Ag 3d5/2 that confirmed the formation of Ag NPs. Ag NPs were spherical in shape with an average size of 30 nm as revealed by HR-TEM and FE-SEM techniques. EDX studies verified the high purity of Ag NPs with silver 46.96%, carbon 16.35%, oxygen 16.22%, nitrogen 20.25% and sulphur 0.21%. LC-MS analysis of plant extract confirmed the qualitative presence of alkaloids, tannins, flavonoids, phenols, and carbohydrates. Prepared Ag NPs showed good photocatalytic activity towards degradation of 4-Amniopyridine with 61% degradation efficiency at optimum conditions in 2 h of reaction time under visible light. The ten intermediates were found within the mass number of 0-450. Ag NPs synthesised using bio-extract have also shown good inactivation against Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) bacteria due to the availability of free radicals.

Structural and functional studies of a snake venom phospholipase A2-like protein complexed to an inhibitor from Tabernaemontana catharinensis.

Snake envenomation is an ongoing global health problem and tropical neglected disease that afflicts millions of people each year. The only specific treatment, antivenom, has several limitations that affects its proper distribution to the victims and its efficacy against local effects, such as myonecrosis. The main responsible for this consequence are the phospholipases A2 (PLA2) and PLA2-like proteins, such as BthTX-I from Bothrops jararacussu. Folk medicine resorts to plants such as Tabernaemontana catharinensis to palliate these and other snakebite effects. Here, we evaluated the effect of its root bark extract and one of its isolated compounds, 12-methoxy-4-methyl-voachalotine (MMV), against the in vitro paralysis and muscle damage induced by BthTX-I. Secondary and quaternary structures of BthTX-I were not modified by the interaction with MMV. Instead, this compound interacted in an unprecedented way with the region inside the toxin hydrophobic channel and promoted a structural change in Val31, loop 58-71 and Membrane Disruption Site. Thus, we hypothesize that MMV inhibits PLA2-like proteins by preventing entrance of fatty acid into the hydrophobic channel. These data may explain the traditional use of T. catharinensis extract and confirm MMV as a promising candidate to complement antivenom or a structural guide to develop more effective inhibitors.

Chemistry of the chippiine/dippinine/tronocarpine class of indole alkaloids.

The chippiines/dippinines/tronocarpine are a family of biologically and structurally interesting polycyclic tryptamine-derived indole alkaloids isolated from the leaf and bark extracts of plants belonging to the Tabernaemontana genus. To date, 14 members of this family have been isolated and characterized. This review discusses the isolation, structure determination, biological activity, and proposed biosynthesis of these metabolites. In addition, synthetic studies on the alkaloids are described including approaches to tronocarpine and dippinine B core intermediates and total syntheses of (+)-dippinine B and (+)-tronocarpine.

Biosynthesis of an Anti-Addiction Agent from the Iboga Plant.

(-)-Ibogaine and (-)-voacangine are plant derived psychoactives that show promise as treatments for opioid addiction. However, these compounds are produced by hard to source plants, making these chemicals difficult for broad-scale use. Here we report the complete biosynthesis of (-)-voacangine, and de-esterified voacangine, which is converted to (-)-ibogaine by heating, enabling biocatalytic production of these compounds. Notably, (-)-ibogaine and (-)-voacangine are of the opposite enantiomeric configuration compared to the other major alkaloids found in this natural product class. Therefore, this discovery provides insight into enantioselective enzymatic formal Diels-Alder reactions.

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.

Cytochrome P450 and O-methyltransferase catalyze the final steps in the biosynthesis of the anti-addictive alkaloid ibogaine from Tabernanthe iboga.

Monoterpenoid indole alkaloids are a large (∼3000 members) and structurally diverse class of metabolites restricted to a limited number of plant families in the order Gentianales. Tabernanthe iboga or iboga (Apocynaceae) is native to western equatorial Africa and has been used in traditional medicine for centuries. Howard Lotsof is credited with bringing iboga to the attention of Western medicine through his accidental discovery that iboga can alleviate opioid withdrawal symptoms. Since this observation, iboga has been investigated for its use in the general management of addiction. We were interested in elucidating ibogaine biosynthesis to understand the unique reaction steps en route to ibogaine. Furthermore, because ibogaine is currently sourced from plant material, these studies may help improve the ibogaine supply chain through synthetic biology approaches. Here, we used next-generation sequencing to generate the first iboga transcriptome and leveraged homology-guided gene discovery to identify the penultimate hydroxylase and final O-methyltransferase steps in ibogaine biosynthesis, herein named ibogamine 10-hydroxylase (I10H) and noribogaine-10-O-methyltransferase (N10OMT). Heterologous expression in Saccharomyces cerevisiae (I10H) or Escherichia coli (N10OMT) and incubation with putative precursors, along with HPLC-MS analysis, confirmed the predicted activities of both enzymes. Moreover, high expression levels of their transcripts were detected in ibogaine-accumulating plant tissues. These discoveries coupled with our publicly available iboga transcriptome will contribute to additional gene discovery efforts and could lead to the stabilization of the global ibogaine supply chain and to the development of ibogaine as a treatment for addiction.

Solution of the multistep pathway for assembly of corynanthean, strychnos, iboga, and aspidosperma monoterpenoid indole alkaloids from 19E-geissoschizine.

Monoterpenoid indole alkaloids (MIAs) possess a diversity of alkaloid skeletons whose biosynthesis is poorly understood. A bioinformatic search of candidate genes, combined with their virus-induced gene silencing, targeted MIA profiling and in vitro/in vivo pathway reconstitution identified and functionally characterized six genes as well as a seventh enzyme reaction required for the conversion of 19E-geissoschizine to tabersonine and catharanthine. The involvement of pathway intermediates in the formation of four MIA skeletons is described, and the role of stemmadenine-O-acetylation in providing necessary reactive substrates for the formation of iboga and aspidosperma MIAs is described. The results enable the assembly of complex dimeric MIAs used in cancer chemotherapy and open the way to production of many other biologically active MIAs that are not easily available from nature.

Eco-friendly preparation of zinc oxide nanoparticles using Tabernaemontana divaricata and its photocatalytic and antimicrobial activity.

The present work reports the green synthesis of Zinc Oxide Nanoparticles (ZnO NPs) using aqueous Tabernaemontana divaricata green leaf extract. ZnO NPs have been characterized by X-ray diffraction (XRD), Ultra Violet-Visible (UV-Vis) studies, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Fourier Transform-Infra Red (FT-IR) analysis. XRD pattern analysis confirms the presence of pure hexagonal wurtzite crystalline structure of ZnO. The TEM images reveal the formation of spherical shape ZnO NPs with the sizes ranging from 20 to 50 nm. The FT-IR analysis suggests that the obtained ZnO NPs have been stabilized through the interactions of steroids, terpenoids, flavonoids, phenyl propanoids, phenolic acids and enzymes present in the leaf extract. Mechanism for the formation of ZnO NPs using Tabernaemontana divaricata as bioactive compound is proposed. As prepared ZnO NPs reveals antibacterial activity against three bacterial strains, Salmonella paratyphi, Escherichia coli and Staphylococcus aureus. The ZnO NPs shows higher antibacterial activity against S. aureus and E. coli and lesser antibacterial activity against S. paratyphi compared to the standard pharmaceutical formulation. Photocatalytic activity of synthesized ZnO NPs was analyzed for methylene blue (MB) dye degradation with sunlight. Almost complete degradation of dye occurred in 90 min. This nano-ZnO, prepared by eco-friendly method will be much useful for dye removal and bacterial decontamination.

Efeitos da fração acetato de etila de Tabernaemontana catharinensis sobre respostas glicêmicas e estresse oxidativo de Rattus norvegicus diabéticos / Effects of tabernaemontana catharinensis ethyl acetate fraction on glycemic responses and oxidative stress

OBJECTIVE: To evaluate the Tabernaemontana catharinensis ethyl acetate fraction hypoglycemic and antioxidant activity through the peripheral glycemic dosage and enzymatic tests. Methods: Male rats were divided into 6 groups: control, diabetic control, control extract 50, diabetic extract 50, control extract 80, diabetic extract 80. In diabetic group animals alloxan (150mg/Kg) was administered to induce Diabetes Mellitus. The animals were beheaded following 15 days of treatment with extract or distilled water and the blood was collected in order to perform oxidative stress tests. Results: The diabetic control group showed high levels of glucose, increased levels of thiobarbituric acid and superoxide dismutase activity, and decreased activity of catalase and glutathione peroxidase enzymes. The diabetic animals that received 50mg/Kg and 80mg/Kg of extract showed a decrease in thiobarbituric acid levels and an increase of glutathione peroxidase activity when compared to the diabetic control group. It was observed that only animals treated with 80mg/Kg of extract had positive results regarding superoxide dismutase. Conclusions: The Tabernaemontana catharinensis ethyl acetate fraction when orally administered for 14 consecutive days at doses of 50mg/Kg and 80mg/Kg reduces the oxidative stress induced by alloxan administration

OBJETIVO: Avaliar a ação hipoglicemiante e antioxidante da fração acetato de etila do extrato de Tabernaemontana catharinensis através da dosagem glicêmica periférica e testes enzimáticos. MÉTODOS: Ratos machos foram divididos em seis grupos: controle, controle diabético, controle extrato 50, diabético extrato 50, controle extrato 80, diabético extrato 80. Nos animais dos grupos diabéticos foi induzida Diabetes Mellitus pela administração de 150mg/Kg de aloxana. Após 15 dias de tratamento com a fração acetato de etila de Tabernaemontana catharinensis ou água destilada, os animais foram decapitados e o sangue foi coletado para realização dos testes de estresse oxidativo. RESULTADOS: O grupo controle diabético apresentou níveis elevados de glicose, aumento dos níveis de ácido tiobarbitúrico e atividade da superóxido dismutase, e diminuição da atividade das enzimas catalase e glutationa peroxidase. Os animais dos grupos diabéticos tratados com 50 e 80mg/Kg do extrato apresentaram redução nos níveis de ácido tiobarbitúrico e aumento da atividade de glutationa peroxidase quando comparado ao grupo controle diabético. Apenas os animais que receberam o extrato na dose de 80mg/Kg obtiveram resultados positivos em relação ao superóxido dismutase. CONCLUSÕES: A fração acetato de etila de Tabernaemontana catharinensis, quando administrada por 14 dias consecutivos, via oral, nas doses de 50 e 80mg/Kg, promove redução nos níveis de estresse oxidativo gerado pela administração de aloxana

Reversible acetylcholinesterase inhibitory effect of Tabernaemontana divaricata extract on synaptic transmission in rat CA1 hippocampus.

BACKGROUND & OBJECTIVES: Acetylcholinesterase inhibitors (AChE-Is) are used for the treatment of Alzheimer's disease (AD). These drugs including galanthamine have been shown to modulate synaptic activity in hippocampus and improve memory processes. Although Tabernaemontana divaricata extract (TDE) has been used as traditional medicine for various pharmacological effects, its effect in enhancing cholinergic activity provides additional benefit to its known effects. We investigated whether TDE can modulate the synaptic function in hippocampus and compared its effects to those of galanthamine. METHODS: Hippocampal slices were prepared from male wistar rats, functional effects of TDE were characterized by using pharmacological tools and extracellular recordings of field excitatory postsynaptic potentials (fEPSPs). RESULTS: TDE significantly reduced fEPSPs. The fEPSPs reduction was prevented by atropine, but not pancuronium. These TDE effects were similar to those of galanthamine. INTERPRETATION & CONCLUSIONS: Our findings indicate that TDE can effectively modulate synaptic responses in the hippocampus similar to galanthamine, suggesting that this traditional medicine could be beneficial in ageing with ACh deprivation in the brain.

Induction of apoptosis in HuH-7 cancer cells by monoterpene and beta-carboline indole alkaloids isolated from the leaves of Tabernaemontana elegans.

Three known (1-3) and a novel (4) monoterpene indole alkaloids have been isolated from the methanol extract of leaves of Tabernaemontana elegans and their structures were elucidated by a series of spectroscopic experiments, involving NMR, MS, UV, and IR techniques. The isolated monoterpene indole alkaloids along with previously described beta-carbolines (5-7) from the same specimen were studied for their apoptosis induction activity in human hepatoma HuH-7 cells. Methodology for apoptosis induction studies included cell viability assays, nuclear morphology assessments, and general caspase-3-like activity assays. The monoterpene indole alkaloids, tabernaemontanine (1) and vobasine (3) showed the most promising apoptosis induction profile in HuH-7 cells.

A thermostable cysteine protease precursor from a tropical plant contains an unusual C-terminal propeptide: cDNA cloning, sequence comparison and molecular modeling studies.

We report here the cloning and characterization of the entire cDNA of a papain-like cysteine protease from a tropical flowering plant. The 1098-bp ORF of the cDNA codify a protease precursor having a signal peptide of 19 amino acids, a cathepsin-L like N-terminal proregion of 114 amino acids, a mature enzyme part of 208 amino acids and a C-terminal proregion of 24 amino acids. The derived amino acid sequence of the mature part tallies with the thermostable cysteine protease Ervatamin-C--as was aimed at. The C-terminal proregion of the protease has altogether a different sequence pattern not observed in other members of the family and it contains a negatively charged helical zone. The three-dimensional model of the precursor, based on the homology modeling and X-ray structure, shows that the extended peptide stretch region of the N-terminal propeptide, covering the interdomain cleft, contains protruding side chains of positively charged residues. This study also indicates that the negatively charged zone of C-terminal propeptide may interact with the positively charged zone of the N-terminal propeptide in a cooperative manner in the maturation process of this enzyme.

Quantification of metabolic flux in plant secondary metabolism by a biogenetic organizational approach.

Metabolic engineering represents a promising approach to enhance the yield of valuable natural products from plants. A method to quantify flux through metabolite measurements is necessary for the analysis of native and modified pathways. Rather than focusing only on the accumulation of the final products, analyzing a wide range of secondary metabolites has significant advantages. We propose a model that organizes the flux analysis by grouping metabolites of similar biosynthetic origin. To this end, we have quantified temporal profiles of metabolites from several branches of the indole alkaloid pathway in Catharanthus roseus hairy root cultures. By analyzing these data, we are able to examine the distribution of flux around key branchpoints. Furthermore, this analysis provides crucial information such as an estimate of total flux to secondary metabolism.