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1.
Int J Mol Sci ; 22(1)2020 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-33374919

RESUMO

A sesquiterpene lactone, thapsigargin, is a phytochemical found in the roots and fruits of Mediterranean plants from Thapsia L. species that have been used for centuries in folk medicine to treat rheumatic pain, lung diseases, and female infertility. More recently thapsigargin was found to be a potent cytotoxin that induces apoptosis by inhibiting the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump, which is necessary for cellular viability. This biological activity encouraged studies on the use of thapsigargin as a novel antineoplastic agent, which were, however, hampered due to high toxicity of this compound to normal cells. In this review, we summarized the recent knowledge on the biological activity and molecular mechanisms of thapsigargin action and advances in the synthesis of less-toxic thapsigargin derivatives that are being developed as novel anticancer drugs.


Assuntos
Antineoplásicos/uso terapêutico , Medicina Tradicional/métodos , Neoplasias/tratamento farmacológico , Thapsia/química , Tapsigargina/uso terapêutico , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Tapsigargina/química , Resposta a Proteínas não Dobradas/efeitos dos fármacos
2.
Drug Des Devel Ther ; 13: 2787-2798, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496655

RESUMO

Objective: Thapsigargin (TG) is a natural product that exists in most parts of the plant Thapsia garganica L. and possesses potential anticancer activities against variety tumor cell lines. TG induces endoplasmic reticulum (ER) stress and apoptosis by inhibiting cancer growth. However, the antineoplastic effect of TG in human adrenocortical carcinoma (ACC) cells is still unknown. Methods: In this study, two human ACC cell lines including SW-13 and NCI-H295R were employed to explore the potential role of TG in ACC. A mouse xenograft model of SW-13 cells was established to verify the role of TG in vivo. The cell viability was tested using Cell Counting Kit-8 and Transwell assays. Flow cytometry and Hoechst 33,258 staining were employed to analyze cell apoptosis. RT-qPCR and Western blot (WB) were performed to explore the underlying mechanism of TG-induced apoptosis in ACC cells. Results: The results indicated that TG dose-dependently inhibited proliferation, migration and invasion in human ACC cells. TG significantly increased the mitochondrial rate of apoptosis and ER stress activity in ACC cells and suppressed ACC xenograft growth in vivo. In addition, the expression of Jun N-terminal kinase (JNK) signaling-related genes and proteins was upregulated by the treatment with TG. Conclusion: Our findings suggest that TG inhibits the viability of ACC cells by inducing apoptosis through the activation of JNK signaling. Thus, TG is expected to be a potential candidate for the treatment of ACC.


Assuntos
Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Carcinoma Adrenocortical/tratamento farmacológico , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Tapsigargina/farmacologia , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/metabolismo , Carcinoma Adrenocortical/patologia , Animais , Antineoplásicos Fitogênicos/química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Thapsia/química , Tapsigargina/química , Células Tumorais Cultivadas
3.
Artigo em Inglês | MEDLINE | ID: mdl-30706829

RESUMO

BACKGROUND AND OBJECTIVE: Lipase inhibitors have gained great interest because they could help in the therapy of many diseases, however, unfortunately, only a few drugs are currently available on the market. Therefore, the aim of this work was to evaluate for the first time the lipase inhibition effect of Thapsia garganica extracts from seeds, leaves and roots. METHODS: Polyphenols and flavonoids contents were determined using spectrophotometric method. Inhibitory activity of ethyl acetate extracts from seeds, leaves and roots of T. garganica against Candida rugosa lipase was determined. To uncover the active constituents responsible for this anti-lipase activity, further investigations were performed by employing theoretical docking simulations, using AutoDock Vina program to discuss the nature of interactions and the inhibition mechanism by major bioactive compounds synthesized by this plant. RESULTS: Seeds, leaves and roots extracts of T. garganica showed appreciable contents of polyphenols and flavonoids which is most in seeds extract with 2.90±0.02mg GAE/gdw and 1.53±0.05mg QE/gdw, respectively. Hence, their inhibitory activities against Candida rugosa lipase were determined as IC50 of 1.19mg/ml, 1.96mg/ml and 1.87mg/ml, respectively. Docking simulations have shown that nortribolid and tribolid are best inhibitors for both lipases (Candida rugosa and human pancreatic lipases). CONCLUSION: Testing the anti-lipase activity of the ethyl acetate extracts of T. garganica revealed a potent lipase inhibition activity, which suggests the use of these molecules as anti-obesity drugs.


Assuntos
Lipase/antagonistas & inibidores , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Thapsia/química , Fármacos Antiobesidade/química , Fármacos Antiobesidade/isolamento & purificação , Fármacos Antiobesidade/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Bioensaio , Flavonoides/química , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Humanos , Técnicas In Vitro , Lipase/química , Lipase/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Extratos Vegetais/química , Folhas de Planta/química , Raízes de Plantas/química , Polifenóis/química , Polifenóis/isolamento & purificação , Polifenóis/farmacologia , Sementes/química
4.
Phytochemistry ; 157: 168-174, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30412824

RESUMO

The genus Thapsia produces a wide variety of sesquiterpenoids. The Mediterranean plant Thapsia laciniata Rouy is known to have a product profile that differs from several other species in the genus. Thus, the biosynthesis of sesquiterpenoids in Thapsia laciniata Rouy was investigated. Here we describe three terpene synthases, TlTPS820, TlTPS509 and TlTPS18983. TlTPS18983 is a multi-product enzyme with farnesene as the major product, while TlTPS509 produces guaiol and bulnesol along with other major and several minor unknown products. TlTPS820 is orthologous to TgTPS2 from Thapsia garganica L. and is an epikunzeaol synthase. TgCYP76AE2 from Thapsia garganica performs a triple hydroxylation of epikunzeaol at C-12 to make dihydrocostunolide. It was therefore investigated if the cytochrome P450, TlCYP76AE4 was able to use epikunzeaol as a substrate. It was found that TlCYP76AE4 hydroxylates epikunzeaol at C-8 to yield tovarol instead of dihydrocostunolide.


Assuntos
Sesquiterpenos/metabolismo , Thapsia/metabolismo , Biocatálise , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxilação , Sesquiterpenos/química , Thapsia/enzimologia
5.
J Ethnopharmacol ; 211: 340-347, 2018 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-28993279

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Thapsia garganica, is a herbal medicine traditionally used as diuretic, emetic and purgative. It is also used as anti-scorpion venom in Morocco; however, its protective effects against scorpion venom remain elusive. AIM OF THE STUDY: The present study was undertaken to evaluate anti-venom activity of T. garganica in vivo through histological and biochemical studies. MATERIALS AND METHODS: Methanolic leaves extract of T. garganica was evaluated for anti-venom activity against buthus. occitanus under in vivo conditions. Histopathological and biochemical changes in envenomed and treated mice were also examined. Phytochemical screening was conducted to estimate the major constituents whereas DPPH, ß -Carotene-linoleic acid and reducing power assays were performed to evaluate the anti-oxidant activity of T. garganica extract. RESULTS: Methanolic leaves extract of T. garganica (2g/kg) increased the survival time (> 18h) of mice injected with lethal doses of B. occitanus venom, with remarkable recovery of histology damage. Furthermore T. garganica induced a significant decreased of biochemical markers of kidney, liver and heart function. Phytochemistry screening revealed the presence of phenolic compounds, flavonoids, tannins and steroids/terpenoids, which might explain the bioactivity of the extract. It was also shown that the extract has an exceptionally high antioxidant activity compared to well-known antioxidants used as standards. CONCLUSION: The present study provides strong evidence that support the use of T. garganica as anti-scorpion venom in traditional medicine in Morocco. However, additional studies are required to isolate and identify the metabolites responsible for the activity.


Assuntos
Antioxidantes/uso terapêutico , Antivenenos/uso terapêutico , Extratos Vegetais/uso terapêutico , Venenos de Escorpião/toxicidade , Thapsia , Animais , Antioxidantes/análise , Antioxidantes/toxicidade , Antivenenos/análise , Antivenenos/toxicidade , Rim/efeitos dos fármacos , Rim/patologia , Dose Letal Mediana , Fígado/efeitos dos fármacos , Fígado/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Medicina Tradicional , Metanol/química , Camundongos , Marrocos , Miocárdio/patologia , Extratos Vegetais/análise , Extratos Vegetais/toxicidade , Folhas de Planta , Solventes/química
6.
Molecules ; 22(10)2017 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-28937623

RESUMO

The composition of the essential oil (EO) of Thapsia villosa (Apiaceae), isolated by hydrodistillation from the plant's aerial parts, was analysed by GC and GC-MS. Antifungal activity of the EO and its main components, limonene (57.5%) and methyleugenol (35.9%), were evaluated against clinically relevant yeasts (Candida spp., Cryptococcus neoformans and Malassezia furfur) and moulds (Aspergillus spp. and dermatophytes). Minimum inhibitory concentrations (MICs) were measured according to the broth macrodilution protocols by Clinical and Laboratory Standards Institute (CLSI). The EO, limonene and methyleugenol displayed low MIC and MFC (minimum fungicidal concentration) values against Candida spp., Cryptococcus neoformans, dermatophytes, and Aspergillus spp. Regarding Candida species, an inhibition of yeast-mycelium transition was demonstrated at sub-inhibitory concentrations of the EO (MIC/128; 0.01 µL/mL) and their major compounds in Candida albicans. Fluconazole does not show this activity, and the combination with low concentrations of EO could associate a supplementary target for the antifungal activity. The association of fluconazole with T. villosa oil does not show antagonism, but the combination limonene/fluconazole displays synergism. The fungistatic and fungicidal activities revealed by T. villosa EO and its main compounds, associated with their low haemolytic activity, confirm their potential antimicrobial interest against fungal species often associated with human mycoses.


Assuntos
Antifúngicos/farmacologia , Arthrodermataceae/efeitos dos fármacos , Aspergillus/efeitos dos fármacos , Candida/efeitos dos fármacos , Malassezia/efeitos dos fármacos , Óleos Voláteis/farmacologia , Thapsia/química , Testes de Sensibilidade Microbiana , Óleos Voláteis/química
8.
Plant Physiol ; 174(1): 56-72, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28275147

RESUMO

The Mediterranean plant Thapsia garganica (dicot, Apiaceae), also known as deadly carrot, produces the highly toxic compound thapsigargin. This compound is a potent inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase calcium pump in mammals and is of industrial importance as the active moiety of the anticancer drug mipsagargin, currently in clinical trials. Knowledge of thapsigargin in planta storage and biosynthesis has been limited. Here, we present the putative second step in thapsigargin biosynthesis, by showing that the cytochrome P450 TgCYP76AE2, transiently expressed in Nicotiana benthamiana, converts epikunzeaol into epidihydrocostunolide. Furthermore, we show that thapsigargin is likely to be stored in secretory ducts in the roots. Transcripts from TgTPS2 (epikunzeaol synthase) and TgCYP76AE2 in roots were found only in the epithelial cells lining these secretory ducts. This emphasizes the involvement of these cells in the biosynthesis of thapsigargin. This study paves the way for further studies of thapsigargin biosynthesis.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Plantas/metabolismo , Thapsia/metabolismo , Tapsigargina/metabolismo , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/genética , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Modelos Químicos , Estrutura Molecular , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Thapsia/citologia , Thapsia/genética , Tapsigargina/síntese química , Tabaco/genética , Tabaco/metabolismo
9.
Nat Prod Res ; 31(13): 1583-1588, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28111971

RESUMO

Polyphenols, flavonoids and condensed tannins contents, as well as the antioxidant activity of the ethanolic and aqueous extracts obtained from aerial parts of 10 wild Tunisian plants, have been determined. Extracts showed appreciable levels of polyphenols and flavonoids, which reached 215.16 mg GAE g-1 DW in Lavandula stoechas ethanolic extract, and 49.12 mg RE g-1 DW in Thapsia garganica aqueous extract. The majority of tested extracts exhibited low total condensed tannins content, except for Rhus tripartitum and Periploca laevigata. The antioxidant activity tests showed great activity, especially for R. tripartitum and Lavandula multifida (IC50 = 5.16 and 5.1 µg mL-1, respectively). Canonical Correspondence Analysis revealed clear groupings of species according to the solvent used.


Assuntos
Antioxidantes , Flavonoides/análise , Compostos Fitoquímicos , Extratos Vegetais/química , Plantas Medicinais/química , Polifenóis/análise , Antioxidantes/química , Etanol , Lavandula/química , Fenóis/análise , Proantocianidinas/análise , Rhus/química , Thapsia/química , Tunísia , Água
10.
Nat Prod Res ; 30(9): 1042-52, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26540577

RESUMO

In this study, the chemical composition of the essential oil from flowers and leaves of Thapsia garganica L. collected in Sicily was evaluated by GC and GC-MS. The main components of T. garganica flower oil (T.f.) were chamazulene (58.3%), humulene oxide II (9.0%), tricosane (8.2%) and pentacosane (8.2%). Also the oil from leaves (T.l.) was characterised by high content of chamazulene (49.2%). Other abundant metabolites were 1,4-dimethylazulene (18.5%), (E)-phytol (6.3%) and neophytadiene (5.1%). The comparison with other studied oils of genus Thapsia is discussed. Antimicrobial activity against several micro-organisms, including some ones infesting historical art craft, was also determined.


Assuntos
Anti-Infecciosos/farmacologia , Óleos Voláteis/química , Óleos Voláteis/farmacologia , Thapsia/química , Antibacterianos/farmacologia , Anti-Infecciosos/isolamento & purificação , Antifúngicos/farmacologia , Bactérias/efeitos dos fármacos , Flores/química , Fungos/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Testes de Sensibilidade Microbiana , Folhas de Planta/química , Sicília
11.
J Nat Prod ; 78(6): 1406-14, 2015 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-26078214

RESUMO

The difference in reactivity of the hexaoxygenated natural product thapsigargin (1) and the pentaoxygenated nortrilobolide (3) was compared in order to develop a chemo- and regioselective method for the conversion of nortrilobolide (3) into the natural product 2-acetoxytrilobolide (4). For the first time, a stereoselective synthesis of 2-acetoxytrilobolide (4) is described, which involves two key reactions: the first chemical step was a one-pot substitution-oxidation reaction of an allylic ester into its corresponding α,ß-unsaturated ketone. The second process consisted of a stereoselective α'-acyloxylation of the key intermediate α,ß-unsaturated ketone to afford its corresponding acetoxyketone, which was converted into 2-acetoxytrilobolide (4) in a few steps. This innovative approach would allow the synthesis of a broad library of novel and valuable penta- and hexaoxygenated guaianolides as potential anticancer agents.


Assuntos
Antineoplásicos/síntese química , Azulenos/química , Azulenos/síntese química , Sesquiterpenos de Guaiano/química , Sesquiterpenos de Guaiano/síntese química , Thapsia/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Azulenos/farmacologia , Técnicas de Química Combinatória , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Sesquiterpenos de Guaiano/farmacologia , Estereoisomerismo , Tapsigargina/síntese química , Tapsigargina/química , Tapsigargina/farmacologia
12.
Chem Biodivers ; 12(4): 637-51, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25879507

RESUMO

The chemical composition of the volatile oils obtained from the roots, leaves, flowers, and stems of Thapsia garganica of Tunisian origin was investigated by GC-FID and GC/MS analyses. Sesquiterpene hydrocarbons and oxygenated monoterpenes were predominant in the oils of all plant parts. Bicyclogermacrene (21.59-35.09%) was the main component in the former compound class, whereas geranial (3.31-14.84%) and linalool (0.81-10.9%) were the most prominent ones in the latter compound class. Principal-component (PCA) and hierarchical-cluster (HCA) analyses revealed some common constituents, but also significant variability amongst the oils of the different plant parts. This organ-specific oil composition was discussed in relation to their biological and ecological functions. For the evaluation of the intraspecific chemical variability in T. garganica, the composition of the flower volatile oils from four wild populations was investigated. Bicyclogermacrene, linalool, and geranial were predominant in the oils of three populations, whereas epicubenol, ß-sesquiphellandrene, and cadina-1,4-diene were the most prominent components of the oil of one population. PCA and HCA allowed the separation of the flower oils into three distinct groups, however, no relationship was found between the volatile-oil composition and the geographical distribution and pedoclimatic conditions of the studied populations.


Assuntos
Óleos Voláteis/química , Thapsia/química , Monoterpenos Acíclicos , Biodiversidade , Análise por Conglomerados , Flores/química , Cromatografia Gasosa-Espectrometria de Massas , Monoterpenos/análise , Naftalenos/análise , Óleos Voláteis/análise , Folhas de Planta/química , Óleos Vegetais/química , Raízes de Plantas/química , Caules de Planta/química , Análise de Componente Principal , Sesquiterpenos/análise , Sesquiterpenos de Germacrano/análise , Tunísia
13.
Molecules ; 20(4): 6113-27, 2015 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-25856061

RESUMO

The sesquiterpene lactone thapsigargin is found in the plant Thapsia garganica L., and is one of the major constituents of the roots and fruits of this Mediterranean species. In 1978, the first pharmacological effects of thapsigargin were established and the full structure was elucidated in 1985. Shortly after, the overall mechanism of the Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibition that leads to apoptosis was discovered. Thapsigargin has a potent antagonistic effect on the SERCA and is widely used to study Ca2+-signaling. The effect on SERCA has also been utilized in the treatment of solid tumors. A prodrug has been designed to target the blood vessels of cancer cells; the death of these blood vessels then leads to tumor necrosis. The first clinical trials of this drug were initiated in 2008, and the potent drug is expected to enter the market in the near future under the generic name Mipsagargin (G-202). This review will describe the discovery of the new drug, the on-going elucidation of the biosynthesis of thapsigargin in the plant and attempts to supply the global market with a novel potent anti-cancer drug.


Assuntos
Thapsia/química , Thapsia/fisiologia , Tapsigargina/metabolismo , Tapsigargina/farmacologia , Fermentação , Thapsia/classificação , Tapsigargina/química
14.
Ann Dermatol Venereol ; 141(12): 756-64, 2014 Dec.
Artigo em Francês | MEDLINE | ID: mdl-25433927

RESUMO

BACKGROUND: Acute localized exanthematous pustulosis (ALEP) is a rare and localized variant of acute generalized exanthematous pustulosis (AGEP). Only 15 cases of ALEP have been reported to date in the literature, with all cases following drug administration. We report 6 paediatric cases of ALEP occurring in springtime, with no associated drug administration in any case. PATIENTS AND METHODS: Over the last three years (2011, 2012 and 2013), we observed 6 cases of ALEP in 6 Tunisian children aged between 9 and 14 years. All cases were observed during the spring months. Diagnosis of ALEP was based in all cases on the EuroSCAR criteria and on the definition of ALEP proposed by Prange et al. A drug-related aetiology was ruled out in all cases, with exposure to a specific planned plant (Thapsia garganica) being retained as an aetiological factor in one case. DISCUSSION: Drug administration is the most frequent though not the sole cause of ALEP. The seasonal nature of this dermatosis may suggest other causes, mainly viral infection, plant contact or airborne allergens.


Assuntos
Pustulose Exantematosa Aguda Generalizada/diagnóstico , Dermatoses Faciais/diagnóstico , Pustulose Exantematosa Aguda Generalizada/tratamento farmacológico , Pustulose Exantematosa Aguda Generalizada/epidemiologia , Pustulose Exantematosa Aguda Generalizada/etiologia , Pustulose Exantematosa Aguda Generalizada/patologia , Adolescente , Corticosteroides/uso terapêutico , Antibacterianos/uso terapêutico , Biópsia , Criança , Dermatoses Faciais/tratamento farmacológico , Dermatoses Faciais/epidemiologia , Dermatoses Faciais/etiologia , Dermatoses Faciais/patologia , Feminino , Flores/efeitos adversos , Humanos , Leucócitos/patologia , Masculino , Estações do Ano , Thapsia/efeitos adversos , Tunísia/epidemiologia
15.
Early Sci Med ; 19(5): 424-47, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25581992

RESUMO

Many studies of early modern natural history focus upon observational, empirical techniques. Early moderns also contended with entities which could no longer be observed because they no longer existed. Although it is often assumed that extinction only emerged as a concept in the eighteenth century, the concept of natural loss appeared, often unproblematically, in areas outside natural philosophy. A survey of discussions of the extinct plant silphion across Europe in the sixteenth and seventeenth centuries shows that the possibility of natural loss was well aired. Paper technologies for collecting extinct nature ran parallel to investigations of newly found nature, and thus can place the latter in a new light. Although ideas of natural mutability often drew on ideas of historical or political change rather than philosophical concepts of natural constancy, techniques developed for extinct nature, such as the list of lost things, remained influential for the research agendas of naturalists.


Assuntos
Extinção Biológica , Ferula/fisiologia , História Natural/história , Thapsia/fisiologia , Asteraceae , Europa (Continente) , História do Século XVI , História do Século XVII , Líbia , Plantas Medicinais/fisiologia
16.
Int J Mol Sci ; 14(5): 9080-98, 2013 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-23698765

RESUMO

Thapsia laciniata Rouy (Apiaceae) produces irregular and regular sesquiterpenoids with thapsane and guaiene carbon skeletons, as found in other Apiaceae species. A transcriptomic analysis utilizing Illumina next-generation sequencing enabled the identification of novel genes involved in the biosynthesis of terpenoids in Thapsia. From 66.78 million HQ paired-end reads obtained from T. laciniata roots, 64.58 million were assembled into 76,565 contigs (N50: 1261 bp). Seventeen contigs were annotated as terpene synthases and five of these were predicted to be sesquiterpene synthases. Of the 67 contigs annotated as cytochromes P450, 18 of these are part of the CYP71 clade that primarily performs hydroxylations of specialized metabolites. Three contigs annotated as aldehyde dehydrogenases grouped phylogenetically with the characterized ALDH1 from Artemisia annua and three contigs annotated as alcohol dehydrogenases grouped with the recently described ADH1 from A. annua. ALDH1 and ADH1 were characterized as part of the artemisinin biosynthesis. We have produced a comprehensive EST dataset for T. laciniata roots, which contains a large sample of the T. laciniata transcriptome. These transcriptome data provide the foundation for future research into the molecular basis for terpenoid biosynthesis in Thapsia and on the evolution of terpenoids in Apiaceae.


Assuntos
Biodiversidade , Perfilação da Expressão Gênica/métodos , Terpenos/metabolismo , Thapsia/genética , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Alquil e Aril Transferases , Sequência de Aminoácidos , Ciclo do Ácido Cítrico , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Eletroforese em Gel de Ágar , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Geraniltranstransferase/metabolismo , Dados de Sequência Molecular , Família Multigênica , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de RNA , Sesquiterpenos/metabolismo , Terpenos/química , Thapsia/enzimologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-23537742

RESUMO

Thapsigargin (Tg) is a selective and irreversible inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-dependent pump at subnanomolecular concentrations. As such, it has become a powerful tool in the study of Ca(2+) signaling pathway. Purification of Tg from Thapsia species requires repeated chromatographic steps with normal-phase alumina or silica and reverse phase chromatography. We thus developed an innovative procedure coupling high pressure automatized extraction with centrifugal partition chromatography allowing a fast and safe large-scale isolation of highly pure Tg, in two steps from Thapsia garganica L. roots. Comparison of influence of extraction procedures, storage conditions and harvesting areas on Tg content in different Algerian specimens of Thapsia garganica L. roots has been precised by mean of HPLC quantification procedure. Highest Tg content were found in the fresh material of the sample from Setif area.


Assuntos
Centrifugação/métodos , Cromatografia Líquida de Alta Pressão/métodos , Inibidores Enzimáticos/farmacologia , Raízes de Plantas/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Thapsia/química , Inibidores Enzimáticos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Espectrofotometria Ultravioleta
19.
Biochem J ; 448(2): 261-71, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22938155

RESUMO

Thapsigargin is a major terpenoid constituent of Thapsia garganica root. Owing to its potent antagonistic effect on the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, thapsigargin has been widely used to study Ca2+ signalling and is also a potential drug for prostate cancer. Despite its importance, thapsigargin biosynthesis in T. garganica remains unknown. In order to decipher thapsigargin biosynthesis, deep transcript sequencing (454 and Illumina) of the T. garganica root was performed, and two terpene synthases (TgTPS1/2) were identified. Functional characterization of their encoded enzymes in a metabolically engineered yeast revealed that TgTPS1 synthesized δ-cadinene, whereas TgTPS2 produced ten distinct terpenoids. However, cultivation of the TgTPS2-expressing yeast in pH-maintained conditions (pH 6-7) yielded one major oxygenated sesquiterpenoid, suggesting that formation of multiple terpenoids was caused by acidity. The major terpene product from TgTPS2 was identified as 6ß-hydroxygermacra-1(10),4-diene (kunzeaol) by mass-fragmentation pattern, retention index, the nature of its acid-induced degradation and NMR. Also, recombinant TgTPS2 efficiently catalysed the synthesis of kunzeaol in vitro from farnesyl diphosphate with a Km of 2.6 µM and a kcat of 0.03 s-1. The present paper is the first report of a kunzeaol synthase, and a mechanism for the transformation of kunzeaol into the thapsigargin backbone is proposed.


Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas de Plantas/metabolismo , Sesquiterpenos/metabolismo , Thapsia/enzimologia , Tapsigargina/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , DNA de Plantas/genética , Cromatografia Gasosa-Espectrometria de Massas , Expressão Gênica , Genes de Plantas , Cinética , Modelos Biológicos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sesquiterpenos/química , Thapsia/genética , Thapsia/metabolismo , Tapsigargina/química
20.
J Pharm Pharmacol ; 64(6): 855-61, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22571264

RESUMO

OBJECTIVES: The aim of this study was to determine whether diacylglycerol kinase (DGK) is involved in transplasmalemmal Ca²âº influx of platelets. METHODS: Effects of R59949, an inhibitor of diacylglycerol kinase, on intracellular Ca²âº concentration ([Ca²âº](i) ) and mRNA expression of DGK isozymes were investigated using washed human platelet suspensions. KEY FINDINGS: Thrombin-induced increase in [Ca²âº](i) was significantly inhibited by pretreatment of platelets with R59949, while thapsigargin-induced increase in [Ca²âº](i) was comparable in platelets with and without R59949 pretreatment. Thapsigargin-induced increase in [Ca²âº](i) was markedly attenuated in the presence of SKF-96365. In the presence of SKF-96365, thrombin-induced increase in [Ca²âº](i) was significantly attenuated, and additional treatment with R59949 caused a further decrease in [Ca²âº](i) . Pretreatment of platelets with 1-butanol significantly attenuated thrombin-induced increase in [Ca²âº](i) , while thrombin-induced increase in [Ca²âº](i) was augmented in the presence of propranolol. mRNA expression of DGK-α and DGK-γ, which are known to be inhibited by R59949, in platelets was confirmed by RT-PCR analysis. CONCLUSIONS: R59949 inhibited a store-depletion-insensitive component of transplasmalemmal Ca²âº entry induced by thrombin, while store-operated Ca²âº entry was not affected by R59949. The results of this study suggest that phosphatidic acid is involved in thrombin-induced Ca²âº influx of platelets.


Assuntos
Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Diacilglicerol Quinase/metabolismo , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Tapsigargina/farmacologia , Trombina/farmacologia , Plaquetas/metabolismo , Butanóis/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Membrana Celular/metabolismo , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/genética , Hemostáticos/farmacologia , Humanos , Imidazóis/farmacologia , Ácidos Fosfatídicos/metabolismo , Extratos Vegetais/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Propranolol/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Thapsia/química
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