RESUMO
Peruvianin-I is a cysteine peptidase (EC 3.4.22) purified from Thevetia peruviana. Previous studies have shown that it is the only germin-like protein (GLP) with proteolytic activity described so far. In this work, the X-ray crystal structure of peruvianin-I was determined to a resolution of 2.15 Å (PDB accession number: 6ORM) and its specific location was evaluated by different assays. Its overall structure shows an arrangement composed of a homohexamer (a trimer of dimers) where each monomer exhibits a typical ß-barrel fold and two glycosylation sites (Asn55 and Asn144). Analysis of its active site confirmed the absence of essential amino acids for typical oxalate oxidase activity of GLPs. Details of the active site and molecular docking results, using a specific cysteine peptidase inhibitor (iodoacetamide), were used to discuss a plausible mechanism for proteolytic activity of peruvianin-I. Histological analyses showed that T. peruviana has articulated anastomosing laticifers, i.e., rows of cells which merge to form continuous tubes throughout its green organs. Moreover, peruvianin-I was detected exclusively in the latex. Because latex peptidases have been described as defensive molecules against insects, we hypothesize that peruvianin-I contributes to protect T. peruviana plants against herbivory.
Assuntos
Glicoproteínas/química , Proteínas de Plantas/química , Thevetia/química , Thevetia/metabolismo , Domínio Catalítico , Simulação de Acoplamento Molecular , Estrutura Quaternária de Proteína , ProteóliseRESUMO
Abstract Thevetia peruviana is an ornamental shrub grown-up in many tropical region of the world. This plant produces secondary metabolites with biological properties of interest for the pharmaceutical industry. The objective was to determine the secondary metabolites profile of callus and cell suspension cultures of T. peruviana and compare them with those from explant (fruit pulp). Extracts in 50% aqueous ethanol and ethyl acetate were prepared. The phytochemical analysis was performed using standard chemical tests and thin layer chromatography. In addition, total phenolic and flavonoids compounds (TPC and TFC), total cardiac glycosides (TCG) and total antioxidant activity (TAA) was determined during the cell suspension growth. Phenolic chemical profile was also analyzed by high performance liquid chromatography (HPLC). Common metabolites (alkaloids, amino acids, antioxidants, cardiac glycosides, leucoanthocyanidins, flavonoids, phenols, sugars and triterpenes) were detected in all samples. The maximum production of extracellular TCG, TPC, TFC and TAA in cells suspensions were at 6-12 days; in contrast, intracellular content was relatively constant during the exponential grown phase (0 to 12-days). HPLC analysis detected one compound with retention time at 11.6 min; this compound was tentatively identified as dihydroquercetin, a flavonoid with anti-cancer properties. These results provide evidence on the utility of the in vitro cell cultures of T. peruviana for valuable pharmaceutical compounds production.
Assuntos
Células Cultivadas , Thevetia/citologia , Compostos Fitoquímicos/biossíntese , Triterpenos , Flavonoides , Cromatografia Líquida de Alta Pressão , Anticarcinógenos , Thevetia/química , Técnicas de Cultura , Compostos Fitoquímicos/análise , AntioxidantesRESUMO
Thevetia peruviana ha sido utilizada en la medicina tradicional mexicana por sus efectos adelgazantes. Para la identificación de las fracciones y los compuestos activos de T. peruviana se utilizó cromatografía en columna abierta y las fracciones obtenidas se evaluaron en ratones con obesidad inducida con glutamato monosódico. Como control positivo se utilizó Orlistat y los tratamientos se administraron oralmente una vez al día durante 8 semanas. La fracción con mayor actividad fue sub-fraccionada y, con la intención de evitar el uso de más ratones, los productos fueron seleccionados por su capacidad para inhibir la adipogénesis en la línea celular 3T3-L1. Los animales tratados con la fracción F-B mostraron un aumento de peso significativamente menor y mantuvieron la sensibilidad a la insulina, así como, niveles significativamente más bajos de colesterol y triglicéridos. El análisis de GC-MS indicó que esta fracción activa está compuesta principalmente por los ácidos palmítico (1), oleico (2) y octadacanoico (3), 2-palmitoil glicerol (4), 2-oleoil glicerol (5) así como la estigmastenona (6).
Thevetia peruviana has been used in Mexican traditional medicine for its slimming effects. Active fractions and compounds from T. peruviana were isolated and identified by means of gravitational open-column chromatography, and the fractions were evaluated on mice with MonoSodium Glutamate-induced obesity. Orlistat was used as positive control, and treatments were administered, orally, once a day for 8 weeks. The fraction with higher activity was sub-fractioned and, with the intention of avoiding using more mice, the fractions were analyzed by evaluating their capability to inhibit adipogenesis in the 3T3-L1 cell line. Animals treated with the F-B fraction revealed a significantly smaller weight increase and maintained adequate insulin sensitivity, and significantly lower blood levels of cholesterol and triglycerides. The active compounds that demonstrated greatest adipogenesis inhibition were palmitic acid (1), oleic acid (2), octadecanoic acid (3), 2-palmitoyl glycerol (4), 2-oleoyl glycerol (5) and stigmastenone (6).
Assuntos
Animais , Camundongos , Fármacos Antiobesidade , Thevetia/química , Glutamato de Sódio , Cromatografia , Medicina Tradicional , MéxicoRESUMO
The Dietary Supplements and Health Education Act (DSHEA), passed by the United States Congress in October of 1994, defines herbal products as nutritional supplements, not medications. This opened the market for diverse products made from plants, including teas, extracts, essential oils, and syrups. Mexico and the United States share an extensive border, where diverse herbal products are available to the public without a medical prescription. Research undertaken in the neighboring cities of Ciudad Juarez, Mexico, and El Paso, Texas, USA, shows the use of herbs is higher in this border area compared to the rest of the United States. A portion of the population is still under the erroneous impression that "natural" products are completely safe to use and therefore lack side effects. We review the dangers of ingesting the toxic seed of Thevetia spp. (family Apocynaceae), commonly known as "yellow oleander" or "codo de fraile," misleadingly advertised on the Internet as an effective and safe dietary supplement for weight loss. Lack of proper quality control regarding herbs generates a great variability in the quantity and quality of the products' content. Herb-drug interactions occur between some herbal products and certain prescription pharmaceuticals. Certain herbs recently introduced into the U.S. market may not have been previously tested adequately for purity, safety, and efficacy. Due to the lack of reliable clinical data regarding the safe use of various herbal products currently available, the public should be made aware regarding the possible health hazards of using certain herbs for therapeutic purposes. The potentially fatal toxicity of yellow oleander seed is confirmed by cases reported from various countries, while the purported benefits of using it for weight loss have not been evaluated by any known clinical trials. For this reason, the use of yellow oleander seed as a dietary supplement should be avoided.
Assuntos
Fármacos Antiobesidade/toxicidade , Suplementos Nutricionais/toxicidade , Sementes/toxicidade , Thevetia/toxicidade , Animais , Fármacos Antiobesidade/economia , Fármacos Antiobesidade/normas , Suplementos Nutricionais/economia , Suplementos Nutricionais/normas , Contaminação de Alimentos/legislação & jurisprudência , Rotulagem de Alimentos/legislação & jurisprudência , Rotulagem de Alimentos/normas , Fraude , Humanos , Internet , Legislação sobre Alimentos , México , Intoxicação por Plantas/etiologia , Intoxicação por Plantas/prevenção & controle , Intoxicação por Plantas/veterinária , Plantas Medicinais/efeitos adversos , Plantas Medicinais/química , Plantas Medicinais/crescimento & desenvolvimento , Plantas Tóxicas/química , Plantas Tóxicas/crescimento & desenvolvimento , Plantas Tóxicas/toxicidade , Sementes/química , Sementes/crescimento & desenvolvimento , Texas , Thevetia/química , Thevetia/crescimento & desenvolvimento , Estados UnidosRESUMO
BACKGROUND: Thevetia peruviana (Pers.) K. Schum or Cascabela peruviana (L.) Lippold (commonly known as ayoyote, codo de fraile, lucky nut, or yellow oleander), native to Mexico and Central America, is a medicinal plant used traditionally to cure diseases like ulcers, scabies, hemorrhoids and dissolve tumors. The purpose of this study was to evaluate the cytotoxic, antiproliferative and apoptotic activity of methanolic extract of T. peruviana fruits on human cancer cell lines. METHODS: The cytotoxic activity of T. peruviana methanolic extract was carried out on human breast, colorectal, prostate and lung cancer cell lines and non-tumorigenic control cells (fibroblast and Vero), using the MTT assay. For proliferation and motility, clonogenic and wound-healing assays were performed. Morphological alterations were monitored by trypan blue exclusion, as well as DNA fragmentation and AO/EB double staining was performed to evaluate apoptosis. The extract was separated using flash chromatography, and the resulting fractions were evaluated on colorectal cancer cells for their cytotoxic activity. The active fractions were further analyzed through mass spectrometry. RESULTS: The T. peruviana methanolic extract exhibited cytotoxic activity on four human cancer cell lines: prostate, breast, colorectal and lung, with values of IC50 1.91 ± 0.76, 5.78 ± 2.12, 6.30 ± 4.45 and 12.04 ± 3.43 µg/mL, respectively. The extract caused a significant reduction of cell motility and colony formation on all evaluated cancer cell lines. In addition, morphological examination displayed cell size reduction, membrane blebbing and detachment of cells, compared to non-treated cancer cell lines. The T. peruviana extract induced apoptotic cell death, which was confirmed by DNA fragmentation and AO/EB double staining. Fractions 4 and 5 showed the most effective cytotoxic activity and their MS analysis revealed the presence of the secondary metabolites: thevetiaflavone and cardiac glycosides. CONCLUSION: T. peruviana extract has potential as natural anti-cancer product with critical effects in the proliferation, motility, and adhesion of human breast and colorectal cancer cells, and apoptosis induction in human prostate and lung cancer cell lines, with minimal effects on non-tumorigenic cell lines.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Glicosídeos Cardíacos/uso terapêutico , Flavonas/uso terapêutico , Neoplasias/tratamento farmacológico , Fitoterapia , Extratos Vegetais/uso terapêutico , Thevetia/química , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Neoplasias da Mama/tratamento farmacológico , Glicosídeos Cardíacos/análise , Glicosídeos Cardíacos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Fragmentação do DNA , Feminino , Flavonas/análise , Flavonas/farmacologia , Frutas , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , México , Extratos Vegetais/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Células VeroRESUMO
MAIN CONCLUSION: The latex from Thevetia peruviana is rich in plant defense proteins, including a 120 kDa cysteine peptidase with structural characteristics similar to germin-like proteins. More than 20,000 plant species produce latex, including Apocynaceae, Sapotaceae, Papaveraceae and Euphorbiaceae. To better understand the physiological role played by latex fluids, a proteomic analysis of Thevetia peruviana (Pers.) Schum latex was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 33 proteins (86 %) were identified, including storage proteins, a peptidase inhibitor, cysteine peptidases, peroxidases and osmotins. An unusual cysteine peptidase, termed peruvianin-I, was purified from the latex by a single chromatographic step involving gel filtration. The enzyme (glycoprotein) was inhibited by E-64 and iodoacetamide and exhibited high specific activity towards azocasein (K m 17.6 µM), with an optimal pH and temperature of 5.0-6.0 and 25-37 °C, respectively. Gel filtration chromatography, two-dimensional gel electrophoresis, and mass spectrometry revealed that peruvianin-I possesses 120 kDa, pI 4.0, and six subunits (20 kDa). A unique N-terminal amino acid sequence was obtained to oligomer and monomers of peruvianin-I (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36). High-resolution images from atomic force microscopy showed the homohexameric structure of peruvianin-I may be organized as a trimer of dimers that form a central channel similar to germin-like proteins. Peruvianin-I exhibited no oxalate oxidase and superoxide dismutase activity or antifungal effects. Peruvianin-I represents the first germin-like protein (GLP) with cysteine peptidase activity, an activity unknown in the GLP family so far.