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.

In vitro antiproliferative activity of alkaloids isolated from Tabernaemontana catharinensis A.DC (Apocynaceae).

In this work, four alkaloids from the stem bark of T. catharinensis were isolated, namely: voacangine (1); ethyl apovincaminate (2); affinisine (3) and voachalotine (4). The alkaloids were tested in vitro for antiproliferative capacity in eight tumor cell lines: U251 (glioma), MCF-7 (breast), NCI-ADR/RES (drug resistant ovary), 786-0 (kidney), NCI-H460 (lung), HT-29 (colon), K562 (leukemia) and PC-3 (prostate) and a non-tumor keratinocyte cell line (HaCat). Antiproliferative activity was observed after 48 hours and results expressed as the concentration needed to induce 50% growth inhibition (GI50) in µM. The chemotherapy drug Doxorubicin was used as a standard. The alkaloid affinisine (3) was the most promising, showing moderate inhibition rates in addition to the cytotoxic and cytocidal effect against all strains tested. It also proved to be a very promising compound, showing high selectivity rates when compared to the non-tumor keratinocyte cell line (HaCat).

Extraction of Phenolic Compounds from Tabernaemontana catharinensis Leaves and Their Effect on Oxidative Stress Markers in Diabetic Rats.

The aim of this study was to evaluate the most effective extraction condition (temperature, solvent type and time) for recovery of high-value phytochemicals present in the Tabernaemontana catharinensis leaves (TC) and to assess their effect on biochemical parameters in streptozotocin-induced diabetic rats. The extraction of phenolic compounds from TC using a factorial design (FD) 2³, high performance liquid chromatography (HPLC), response surface methodology (RSM) and principal component analysis (PCA) were studied. It was found that the optimal conditions for extraction of phenolics were higher temperature (65 °C) and time (60 min) using ethanol as extractor solvent. In this condition of extraction (A8), total phenolic compounds (TPC) and antioxidant activity (AA) were determined. Additionally, this extract was used to evaluate their effect on antioxidant enzyme activities (superoxide dismutase (SOD) and catalase (CAT)) as well as lipid peroxidation (LP) and protein thiols level (PSH) in the liver and kidneys of normal and diabetic rats. As result, T. catharinensis extract presented TPC content of 23.34 mg EAG/g (equivalent gallic acid) and AA of 34.26 µmol Trolox/g. Phenolic acids (ferulic acid and coumaric acid) and flavonoids (quercetin, rutin and pinocembrin) could be recovered and identified by HPLC. This study indicated an important role of the T. catharinensis extract on free radical inactivation and on the antioxidant defense system in diabetic rats. In fact, the use of T. catharinensis extract restored the normal activity of SOD (p < 0.05) and suppressed malondialdehyde levels in liver and kidney tissues. Thus, the T. catharinensis extract, rich in phenolic compounds, can be responsible for the recover the enzymatic changes in the liver and kidney tissues provoked by diabetes in rats. In addition, the lipid peroxidation rate decreased in the diabetic rats treated with T. catharinensis.

Antinociceptive and antioxidant effects of extract enriched with active indole alkaloids from leaves of Tabernaemontana catharinensis A. DC.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological knowledge is important for the identification of active compounds from natural products. Pain may have different aetiologies with complex mechanisms. Tabernaemontana catharinensis A. DC. is well known for indole alkaloids, being used empirically in folk medicine, with antimicrobial and anti-inflammatory as well as antiofidic actions among others. AIM OF THE STUDY: This work aims to evaluate the antinociceptive and antioxidant effect in mice of the alkaloids extract from leaves of Tabernaemontana catharinensis A. DC. (AITc). MATERIALS AND METHODS: The AITc was produced by ultrasound and acid-base extraction, and the chemical composition was evaluated by high resolution mass spectrometry. Male mice (Mus musculus), Swiss, were used for in vivo tests. The AITc was administrated at doses of 1.0, 5.0, and 10.0 mg/kg in acetic acid model, formalin, tail-immersion, hot plate, and open field tests, and compared to saline, morphine, or diazepam controls, depending on the test. The toxicological, biochemical, haemogram and antioxidant effect were evaluated in mouse organs such as liver, brain, kidneys, spleen and stomach. RESULTS: In total, 10 compounds were identified in the AITc, being from the indole alkaloids from the ibogan and corynanthean classes. The extract in doses ranging from 5.0 to 10.0 mg/kg showed an antinociceptive effect for acetic acid, inhibiting by 47.7% and 61.6%. In the same line, reductions of 47.1% (first phase) and 43.6% (second phase) were observed for the 5.0 mg/kg dose in the formalin test. However, tail-immersion and hot plate tests did not show considerable modifications in the latency period, while in the open field test there was an inhibition of only 5.1%. It was observed no differences in NO levels and total antioxidant status of the mice in any of the studie tissues. CONCLUSIONS: The results justify the use of this plant in traditional medicine. in vivo tests indicate that these compounds possess central and peripheral mechanisms of action. This is study that reports the nociceptive action of these alkaloids, also including toxicity tests, which are intended to guarantee the safety of use of extracts of this plant.

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.

Atividade alelopática de extratos aquosos de plantas medicinais na germinação de Lactuca sativa L. e Bidens pilosa L. / Allelopathic activity of aqueous extracts of medicinal plants on the germination of Lactuca sativa L. and Bidens pilosa L.

O potencial alelopático de quatro diferentes extratos de plantas medicinais (Eclipta alba, Gomphrena globosa, Tabernaemontana catharinensis e Tithonia diversifolia) foi avaliado na germinação de Bidens pilosa e Lactuca sativa. Dentre os resultados obtidos no teste de germinação, notou-se um maior poder inibitório do extrato de G. globosa sobre sementes e plântulas de Bidens pilosa e Lactuca sativa independente da concentração utilizada e aumento da atividade inibitória de extratos T. catharinensis e de T. diversifolia com aumento da concentração destes. Quanto ao Índice de Velocidade de Germinação, os dados demonstraram redução do índice devido a elevada mortalidade das plântulas submetidas aos extratos de G. globosa e de T. catharinensis e a germinação tardia das sementes submetidas ao extrato de T. diversifolia. Quanto ao vigor das sementes, as menores porcentagens de sobrevivência ocorreram extratos à 2,5 por cento para B. pilosa e 10 por cento para L. sativa.

The allelopathic potential of four different extracts of medicinal plants (Eclipta alba, Gomphrena globosa, Tabernaemontana catharinensis and Tithonia diversifolia) was evaluated on the germination of Bidens pilosa and Lactuca sativa. The results obtained in the germination test indicated a greater inhibitory activity of G. globosa extract on Bidens pilosa and Lactuca sativa seeds and seedlings, regardlessof the used concentration, and an increase in the inhibitory activity of T. catharinensis and T. diversifolia extracts with an increase in their concentrations. As to Germination Speed Index, the data demonstrated a reduction due to the high mortality of seedlings subjected to G. globosa and T. catharinensis extracts and a late germination of seeds subjected to T. diversifolia extracts. As to the vigor of seeds, the smallest survival percentages occur at 2.5 percent for B. pilosa and at 10 percent for L.sativa.

Avaliação da atividade antimicrobiana in vitro do extrato de Tabernaemontana catharinensis A. DC / Evaluation of in vitro antimicrobial activity of Tabernaemontana catharinensis A. DC. extract

Diversas espécies de Tabernaemontana têm sido estudadas devido a diversidade de alcalóides com atividade farmacológica. O objetivo desse trabalho foi avaliar a capacidade antimicrobiana in vitro do extrato das cascas do caule de Tabernaemontana catharinensis A. DC.em cepas de Staphylococcus aureus e Pseudomonas aeruginosa, microrganismos causadores de diversas infecções. Os testes de susceptibilidade bacteriana foram realizados usando o método de Kirby Bauer, consistindo na difusão em disco do antibiótico em meio de cultivo Mueller Hinton. Os testes de inibição foram realizados com soluções do extrato bruto seco de T. catharinensis dissolvido em etanol 70 por cento (v/v) na concentração 1,0 mg mL-1, que aplicada nos discos de área 20 mm², apresentaram concentração de 0,005 mg mm-2. Como controle negativo, realizou-se ensaios com placas contendo P. aeruginosa, e discos com etanol 70 por cento (v/v), e como controle positivo, discos com os antibióticos ceftriaxona sódica (0,25 mg mm-2 de área do disco), tetraciclina (0,005 mg mm-2) e cefalexina (0,005 mg mm-2). A solução do extrato na concentração de 0,005 mg mm-2 inibiu o Staphylococcus aureus, com diâmetro médio do halo de 0,6 cm. O halo de inibição para o Pseudomonas aeruginosa foi em média 1,2 cm. A tetraciclina, a cefalexina, e o controle negativo (etanol 70 por cento v/v) não demonstraram ação antimicrobiana. O halo de inibição usando ceftriaxona foi em média 2,2 cm para P. aeruginosa e 1,0 cm para Staphylococcus aureus.

Several Tabernaemontana species have been studied due to their several alkaloids with pharmacological activity. The aim of this work was to evaluate the in vitro antimicrobial action of the extract from stem barks of Tabernaemontana catharinensis A. DC. against strains of Staphylococcus aureus and Pseudomonas aeruginosa, microorganisms that cause several infections. Bacterial susceptibility tests were performed by the Kirby-Bauer method, consisting in antibiotic disk diffusion in Mueller Hinton medium. Inhibition tests were performed with solutions of T. catharinensis dry crude extract dissolved in ethanol 70 percent (v/v) at 1.0 mg mL-1, which became 0.005 mg mm-2 when applied to 20 mm² disks. As negative control, assays were carried out in plates containing P. aeruginosa and disks with ethanol 70 percent (v/v). Positive control consisted of disks containing the antibiotics ceftriaxone sodium (0.25 mg mm-2 disk area), tetracycline (0.005 mg mm-2) and cephalexin (0.005 mg mm-2). Extract solution at 0.005 mg mm-2 inhibited Staphylococcus aureus, with 0.6cm halo mean diameter. The inhibition halo for Pseudomonas aeruginosa was on average 1.2 cm. Tetracycline, cephalexin and negative control (ethanol 70 percent v/v) did not show antimicrobial action, whereas ceftriaxone sodium resulted in 2.2 and 1.0cm mean inhibition halo diameters for P. aeruginosa and Staphylococcus aureus, respectively.

Morfo-anatomia da plântula de tabernaemontana catharinensis A. DC. (Apocynaceae) / Morphology and anatomy of the seedling of tabernaemontana catharinensis A. DC. (Apocynaceae)

Tabernaemontana catharinensis, conocida vulgarmente como lechero, es una especie perteneciente a la familia Apocynaceae. El actual estudio se refierea la morfología y la anatomía de la plántula de esta especie. Las semillas colectadas desde varios arboles creciendo en la planicie de la inundación del alto río Paraná, fueron germinadas en una cámara de germinación con una temperatura constante de 300C y mantenidas bajo luz fluorescente continua. El desarrollo de las plántulas ocurrió en un invernadero. La anatomía fue estudiada en plántulas fijadas en FAA 50. Este material fue seccionado transversalmente y las secciones obtenidas fueron teñidas en safranina y azul de astra. La plántula es fanerocotiledonar y epígea y presenta raíz axial, cuello, cotiledones ovados, epicótilo reducido, y eófilos lanceolados semejantes a los metáfilos. Su raíz es tetrarca. El hipocótilo y epicótilo tienen estructura caulinar y presentan crecimiento secundario cuya peridermis es de origen del felógeno en el ámbito epidérmico. Los cotiledones y eófilos son hojas dorsiventrales, con un estrato de parénquima en empalizada y algunas capas de parénquima esponjoso. Los cotiledones tienen un haz vascular colateral en la vena media, mientras en los eófilos el haz es bicolateral.

Tabernaemontana catharinensis is a species belonging to the family Apocynaceae. In this paper, the seedling morphology and anatomy of this species are described and discussed. The seedling development was analyzed until the first eophyll pair’s formation. The seeds were collected from several trees, in fragments of the semideciduous seasonal alluvial and submontaneforest located on the Upper Paraná River floodplain. The seeds were germinated at constant temperatures of 300C, using germination chambers and maintained under continuous fluorescent light. Seedlings development occurred in the greenhouse. The seedlings fixed in FAA 50 were sectioned by microtome and stained with safranin and astra blue. Seedling is phanerocotylar and epigeal with axial root, collet, ovate cotyledons, reduced epicotyl, and lanceolate eophylls similar to the metaphylls. Root is tetrarch. Hypocotyl and epicotyl have stem structure, and they present secondary growth with phellogen of epidermic origin. Cotyledons and eophylls are dorsiventral leaves, with uniseriate palisade parenchyma and some layers of spongy parenchyma. Cotyledons have a collateral vascular bundle in the midrib, while in the eophylls is bicollateral one.