Ultrasound-assisted Extraction of Ursolic Acid from the Flowers of Ixora coccinia Linn (Rubiaceae) and Antiproliferative Activity of Ursolic Acid and Synthesized Derivatives.

BACKGROUND: Ixora coccinea Linn (Rubiaceae) is an evergreen shrub with bright scarlet colored flowers found in several tropical and subtropical countries. It is used as an ornamental and medicinal plant. Phytochemical studies revealed that its major special metabolites are triterpene acids, such as ursolic and oleanolic acid. OBJECTIVE: To evaluate the isolation of ursolic acid (UA) (1) from methanol extracts of I. coccinea flowers through two methodologies, to prepare four derivatives, and to evaluate the cytotoxic effect against six cancer cell lines. MATERIALS AND METHODS: The UA was isolated from vegetal material by percolation at room temperature and by ultrasound-assisted extraction. The preparation of derivatives was performed according to literature methods, and the cytotoxic effects were evaluated using the MTT (3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay. RESULTS: The most efficient extraction was achieved through ultrasound irradiation with a yield of 35% after KOH-impregnated silica in chromatography column. Furthermore, four derivatives (3, 5, 6, 7) of UA were prepared and evaluated, including 1, against two lung cancer (A549 and H460) and four leukemia (K562, Lucena, HL60, and Jurkat) cell lines. Generally, results showed that 1 and 7 were the most active compounds against the assayed cell lines. Also, the cytotoxic effects observed on terpenes 1 and 7 were higher when compared with cisplatin, used as positive control, with the exception of Jurkat cell line. CONCLUSION: The efficiency of such an alternative extraction method led to the principal and abundant active component, 1, of I. coccinea, thus representing a considerable contribution for promising triterpenoid in cancer chemotherapy. SUMMARY: The ultrasound-assisted extraction of Ixora coccinea flowers improved of the ursolic acid isolationMethanolic extract from flowers of I. coccinea provided, by ultrasound irradiation, after KOH-impregnated silica in chromatography column, the ursolic acid in 35% yieldThe ursolic acid and four derivatives were prepared and assayed against two lung cancer and four leukaemia cell linesThe ursolic acid and their 3-oxo-derivative, in general, were more cytotoxic when compared to cisplatin, used as positive control Abbreviations used: MTT: 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide, RP: reverse phase, TLC: thin layer chromatography, KOH: potassium hydroxide, IR: infrared, DMF: dimethylformamide, DMSO: dimethyl sulfoxide, TEA: triethylamine, RT: room temperature, EtOAc: ethyl acetate, MeOH: methanol, i-PrOH: iso-propanol, NMR: nuclear magnetic resonance, MDR: multiple drug resistance, RPMI: Roswell Park Memorial Institute.

Croton cajucara crude extract and isolated terpenes: activity on Trypanosoma cruzi.

Croton cajucara is a plant found in the Amazon region and is known for its medicinal properties. The effects of the methanolic extract of the stem bark of C. cajucara (MCC) and of the isolated terpenes, trans-dehydrocrotonin (t-DCTN) and acetyl aleuritolic acid (AAA), were investigated using four isolates of Trypanosoma cruzi. In assays with trypomastigotes, the extract was more active than the isolated compounds, presenting IC(50) in the range of 10 to 50 µg/mL. The trypanocidal effect of MCC, AAA and benznidazole was significantly higher in the GLT291 and C45 strains, which were recently isolated from wild animals. MCC and AAA caused a dose-dependent inhibition of epimastigote proliferation. In assays using intracellular amastigotes, AAA and MCC reduced the percent of infection and the endocytic index after 96 h of treatment, at concentrations that were non-toxic to the host cells. MCC inhibited the trypanothione reductase pathway in both epimastigotes and trypomastigotes of all the subpopulations. The absence of AAA activity on the trypanothione reductase pathway in epimastigotes of Dm28c suggests heterogeneity of the biochemical profile between this clone and the three strains. Epimastigotes and trypomastigotes (GLT291) were treated for 24 h with MCC or AAA, and both induced alterations of the plasma membrane, while AAA-treated epimastigotes also displayed mitochondrial damage.

Development of a new propolis microemulsion system for topical applications / Desenvolvimento de um novo sistema microemulsionado de própolis para aplicação tópica

Microemulsion systems (MES) offer advantages as drug delivery systems, among them favour drug absorption, being in most case more efficient than other methods in delivering of drug. In this work a new MES was obtained in order to be applied as a pressurized aerosol formulation containing bee propolis ethanolic extract (PEE). For that, pseudoternary phase diagrams were used to characterize the microemulsions boundaries and also to define the Winsor IV microemulsion region of the PEE-MES system containing Tween 80 as surfactant and the cosurfactant ethyl alcohol in small percentage. The obtained results indicated that the best MES was composed by Tween 80 and ethyl alcohol with C/S (cosurfactant/surfactant) ratio equal to 1.0, since it provided a large boundaries in the obtained O/W microemulsion region. This PEE-MES formulation, in which bee propolis consisting as oil phase, is herein designed for topical uses (PEE-MES spraying) in order to treat mouth and throat inflammatory infections. Considering the very large uses of bee propolis in conventional vehicles, MES type of delivery system has to be compatible with achieving the highest drug aim loadings, determined substantially by the specific MES application (drug solubilization in water systems) improving in this case, propolis farmacological aplications. Additionally, PEE-MES antibacterial effect was evidenced and the microemulsion system PEE-MES was also used as newest chemical approach for extraction of bee propolis material from resinous hive.

Sistemas microemulsionados (MES) oferecem inúmeras vantagens como liberadores de fármacos já que favorecem a absorção do princípio ativo e são, em muitos casos, mais eficientes do que outros métodos de liberação de substâncias bioativas. Neste trabalho, um MES foi obtido visando uma formulação para uso tópico, contendo extrato etanólico de própolis (PEE). Neste estudo, Diagramas de fase pseudoternários foram utilizados para caracterização das regiões de microemulsão, bem como para definição da região de Winsor IV do sistema PEE-MES (Tween 80 como tensoativo e álcool etílico como co-tensoativo, em baixo percentual). Os resultados obtidos indicaram que o melhor sistema microemulsionado contém C/S (cotensoativo/tensoativo) na razão 1,0, fornecendo uma microemulsão do tipo O/A com ampla região de Winsor IV. A formulação PEE-MES poderá vir a ser utilizada comercialmente para uso tópico no combate a inflamações de boca e garganta. Considerando o amplo uso de própolis em formulações convencionais, onde sua solubilidade representa um dos maiores problemas, o sistema PEE-MES disponibiliza este produto natural para aplicações farmacológicas, com boa solubilidade em um sistema microemulsionado do tipo O/A. Adicionalmente, o efeito bactericida do sistema PEE-MES foi comprovado e uma nova metodologia de extração de própolis é apresentada.

Potential benefits of the 19-nor-clerodane trans-dehydrocrotonin on the central nervous system / Potenciais benefícios do 19-nor-clerodano trans-desidrocrotonina isolado de Croton cajucara sobre o sistema nervoso central

This study examined the effect of trans-dehydrocrotonin (DCTN), a 19-nor-clerodane diterpene isolated from Croton cajucara Benth (Euphorbiaceae), as analgesic and its effect on the central nervous system (CNS) of rodents using different animal models. The DCTN intraperitoneally exhibited mild analgesic activity on hot-plate test, but exhibited strong antinociceptive activity against acetic acid-induced abdominal writhing and the ED50 was calculated to be 44.88 mg/kg. At higher doses (100 mg/kg) it exhibited mild CNS depressant activities in laboratory animals. Moreover, it has negligible antidepressant activity. After taking consideration of the drug interaction, the DCTN can be used as a potent analgesic agent in case of peripheral algesia, without affecting the CNS.

Neste estudo avaliou-se o efeito analgésico do diterpeno 19-nor-clerodano trans-desidrocrotonina (DCTN) isolado de Croton cajucara Benth (Euphorbiaceae), bem como seu efeito no sistema nervoso central utilizando-se diferentes tipos de modelos de animais roedores. A administração intraperitoneal deste diterpeno, no teste da placa quente, revelou sua atividade analgésica moderada. No entanto, no teste de contrações abdominais desencadeadas por ácido acético, a DCTN apresentou forte atividade antinociceptiva com DE50 de 44,88 mg/kg. Doses elevadas de DCTN (100 mg/kg) apresentaram moderada atividade depressiva do sistema nervoso central (SNC), não tendo sido evidenciado ação antidepressiva. Após algumas considerações da ação de DCTN em algesia periférica, concluiu-se que esta substância pode ser utilizada como um potente agente analgésico, sem afetar o SNC.

1H and 13C NMR assignments for two new cordiaquinones from roots of Cordia leucocephala.

From the roots of Cordia leucocephala (Boraginaceae), two new meroterpenoid naphthoquinones, 6-[10-(12,12-dimethyl-13alpha-hydroxy-16-methenyl-cyclohexyl)ethyl]-1,4-naphthalenedione (cordiaquinone L) and 5-methyl-6-[10-(12,12-dimethyl-13beta-hydroxy-16-methenyl-cyclohexyl)methyl-1,4-naphthalenedione (cordiaquinone M) were isolated. Their structures were elucidated after detailed 1D and 2D NMR (COSY, HSQC, HMBC and NOESY) data analyses and comparison with literature data for analogous compounds.

Encapsulation and release characteristics of DCTN/PLGA microspheres.

In the present study, poly (D,L-lactic-co-glycolic)-acid microspheres containing trans-Dehydrocrotonin (DCTN) were prepared by the double emulsion method. The hypoglycemic activity of DCTN-loaded microspheres was monitored in normal glycemic mice after administration of a daily dose of DCTN (50 mg kg(-1) body weight) for 7 days. Spherical microspheres with two populations of particles with 3.20 +/- 0.10 and 7.60 +/- 0.70 microm mean diameter size microm were observed. The encapsulation efficiency of DCTN was 85.5 +/- 3.9%. The in vitro kinetic profile of DCTN from PLGA-microspheres was initially fast (burst effect of 19.4% at 2 h). Such a burst step was maintained until achieving 35.7+/-2.0% at 7h, followed by a gradual release of DCTN attaining a maximum drug release at 55.7 +/- 2.6% within 30 h. DCTN was able to reduce glucose levels (14.3%) of normal glycemic animals and this effect was improved by its encapsulation into microspheres (26.8%). The optimum glucose levels in the blood of animals treated with DCTN suspension and DCTN-loaded microspheres were 119.21 +/- 19.75 mg dL(-1) at day 5 and 103.08 +/- 18.88 mg dL(-1) at day 7, respectively. DCTN-loaded microspheres are thus offered as a potential delivery system for the treatment of metabolic diseases characterized by hyperglycemia.

Comparative anti-inflammatory and antinociceptive effects of terpenoids and an aqueous extract obtained from Croton cajucara Benth

The 19-nor-clerodane trans-crotonin (CTN) and the triterpene acetyl aleuritolic acid (AAA), isolated from the stem bark of Croton cajucara Benth (Euphorbiaceae), a traditional medicinal plant from Amazon region of Brazil, as well as the aqueous extract (AE) from its stem bark, were submitted to pharmacological screening for anti-inflammatory and antinociceptive activities in animal models. The oral administration of AAA (50 mg/kg), CTN (50 mg/kg) or AE (300 mg/kg) inhibited the acetic acid-induced writhing in mice. The AE, CTN and AAA had shown significant inhibition of carrageenin-induced edema in rats, in all time intervals measured after the injection of the stimulus, with the greatest inhibition at the first hour for AAA (47.7 percent) and the second hour for CTN (54.4 percent). They have also exhibited significant inhibition in the dextran-induced edema 90 minutes after the stimulus: 31.9 percent for CTN and 28.5 percent for AAA. In the histamine-induced edema, the inhibition showed by CTN and AAA were 43.2 percent and 40.5 percent, respectively, 90 minutes after the injection of stimulus. This study extends and supports the popular medicine and folkloric uses of Croton cajucara in the Amazon region of Brazil.

O 19-nor-clerodano trans-crotonina (CTN) e o triterpeno ácido acetil aleuritólico (AAA), isolados das cascas do caule de Croton cajucara Benth (Euphorbiaceae), uma planta tradicional da Região Amazônica do Brasil, bem como o extrato aquoso (EA) das cascas do caule deste Croton, foram submetidos a experimentos farmacológicos utilizando animais, para avaliação das atividades anti-inflamatória e antinociceptiva. A administração oral de AAA (50 mg/kg), CTN (50 mg/kg) ou AE (300 mg/kg) inibiram as contorções em ratos, induzidas por ácido acético. Os terpenóides AAA e CTN, bem como o extrato polar EA, inibiram significativamente o edema de pata em ratos, induzido por carragenina. As inibições foram observadas em todos os intervalos de medições, tendo sido evidenciado melhores inibições para os terpenóides AAA (47,7 por cento, após a primeira hora) e CTN (54,4 por cento, após a segunda hora). Evidenciou-se ainda, após 90 minutos do estímulo, significante inibição no edema induzido por dextrana (31,9 por cento para CTN e 28,5 por cento para AAA) e por histamina (43,2 por cento para CTN e 40,5 por cento para AAA). Estes resultados confirmam o uso popular de Croton cajucara na região Amazônica do Brasil, no combate a inflamações.

Uma revisão das atividades biológicas da trans-desidrocrotonina, um produto natural obtido de Croton cajucara / A review of the biologic activities of trans-dehydrocrotonin, a natural product obtained from Croton cajucara

Croton cajucara Beth (Euphorbiaceae) uma espécie medicinal nativa da região Amazônica do Brasil, onde é vulgarmente conhecida como 'sacaca', representa um recurso terapêutico eficaz no tratamento e cura de várias doenças. O metabólito majoritário trans-desidrocrotonina (DCTN), isolado das cascas do caule desta planta, encontra-se correlacionado com grande parte das propriedades medicinais da sacaca. Este artigo de revisão, descreve os resultados fitoquímicos e farmacológicos que foram realizados com o diterpeno do tipo clerodano DCTN, bem como seus derivados semi-sintéticos. Adicionalmente, apresenta perspectivas para a biodisponibilização deste protótipo de fármaco em nanosistemas.

Croton cajucara Beth (Euphorbiaceae) is a plant found in the Amazonian Region of North Brazil, where it is popularly known as sacaca. The major secondary metabolite, trans-dehydrocrotonin (DCTN) a clerodane-type diterpene, isolated from the stem bark is a chief bioactive compound of Croton cajucara. This review describes results of extensive pharmacological studies of DCTN, as well as its semi-synthetic derivatives, and also presents insights into the use of DCTN as a therapeutic agent and some potential advantages of its incorporation in drug delivery systems.

Composição química do óleo fixo de Croton cajucara e determinação das suas propriedades fungicidas / Chemical composition of the fixed oil of Croton cajucara and its antifungical properties

Este trabalho objetivou a obtenção de óleos fixos (OF) das cascas do caule de Croton cajucara via percolação [seguido de fracionamento em coluna cromatográfica (CC)] e fluido supercrítico (FS, gás CO2), bem como a avaliação das propriedades fungicidas de OF obtido pelo procedimento CC. Os óleos OF-CC e OF-FS apresentaram rendimentos significativamente diferentes; houve maior rendimento (3,14 por cento) de OF obtido no processo convencional (OF-CC), versus 1,2 por cento de OF-FS. A fração F1 após esterificação, bem como as frações não-esterificadas F2, F3 e F4 (obtidas de OF-CC seguido de outro fracionamento em CC) foram analisadas por CG-FID e CG-EM. A identificação da composição química de OF-CC foi feita por comparação com dados da biblioteca Wiley, bem como pela comparação de índices de kovats. A presença de sesquiterpenos foi comprovada na fração F1, onde o a-copaeno (20,1 por cento) e cipereno (21,8 por cento) foram os componentes majoritários (70 por cento de percentual sesquiterpênico total). A fração F2 mostrou sesquiterpenos minoritários mais oxigenados, incluindo o linalol. Na fração F3 foram observados ácidos graxos, misturas de esteróis e os diterpenos bioativos trans-crotonina, cis-cajucarina B e trans-cajucarina B, onde 40 por cento do total desta fração, foi composta de esteróis e diterpenos; e na fração F4 foram observados majoritariamente os isômeros cis e trans-cajucarina B. O efeito biológico de OF-CC foi avaliado no desenvolvimento in vitro dos fungos fitopatogênicos Fusarium oxysporum, Rhizoctonia solani e Sclerotium rolfsii propagados em meio de cultura Batata-dextrose-agar (BDA). A resposta fisiológica, variou de acordo com o tipo de fungo testado, tendo sido observado que o OF-CC (0,2 mg.mL-1) apresentou efeito fungistático no controle micelial dos fungos testados, sendo que o gênero de fungo Fusarium oxysporum foi o que sofreu efeito inibitório mais estável.

This paper focused on the separation of the fixed oil (FO) from the stem bark of Croton cajucara obtained from the conventional approach extraction with organic solvents [followed by chromatography column (CC)] and supercritical fluid extraction (SFE, carried out with CO2) and also, on the antifungical properties of the FO obtained by CC procedure (FO-CC). The FO contents were 3.14 percent for FO-CC versus 1.20 percent in the FO-SFE process. The esterified fraction F1 and non-esterified fractions F2, F3 and F4 obtained from FO-CC after a new CC procedure were analyzed by HRGC-MS. The identification of the chemical composition of FO-CC was made by comparison with MS literature data, computer matching with the Wiley library and by comparison of their kovats indices with the literature. Fraction F1 showed 70 percent of sesquiterpene components, among them a-copaene (20.1 percent) and ciperene (21.8 percent) as major compounds. Fraction F2 was rich in minor oxygenated sesquiterpenes, among them linalool. Meanwhile fraction F3 showed fat acids, steroids and the bioactive clerodane type-diterpene trans-crotonin, cis-cajucarin B (c-CJC-B) and trans-cajucarin B (t-CJC-B). Fractions F3 and F4 showed as major constituents c-CJC-B and t-CJC-B with 40 percent of the total contents. The biological effect of the FO-CC was evaluated in the in vitro development of the phytopatogen fungi such as Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii. Significant inhibitory effect of the tested fungi (at 0.2 mg.mL-1 dosage) were proved.

Absence of mutagenicity in somatic and germ cells of mice submitted to subchronic treatment with an extract of Croton cajucara Benth. ( Euphorbiaceae )

The plant Croton cajucara Benth. (Euphorbiaceae) is a medicinal plant from the Brazilian Amazon where it is commonly known as sacaca. The principal compound isolated from C. cajucara stem-bark extracts is the clerodane-type diterpene trans-dehydrocrotonin (DCTN) which presents several biological activities, including antiulcerogenic, anti-inflammatory, hypoglycemic, antimutagenic and antitumoral activity. However, few studies have been carried out to evaluate the therapeutic potential of raw C. cajucara extracts. We studied mutagenicity and antimutagenicity effects of C. cajucara methanol extract using the micronucleus assay in bone marrow cells and the dominant lethal assay in mice submitted to subchronic treatments. The blood testosterone levels of the mice were also measured to assess the effects of the methanol extract on testes function. Statistical analysis of the data obtained in this study showed no statistically significant mutagenicity attributable to C. cajucara stem-bark extracts, nor did such extracts show antimutagenic activity at the concentrations assessed. The testosterone concentration was normal in all the mice studied.

Cardiovascular effects of trans-dehydrocrotonin, a diterpene from Croton cajucara in rats.

Previous studies have established the gastroprotective, hypoglycemic, and hypolipidemic effects of trans-dehydrocrotonin (t-DCTN), a major diterpene isolated from the Amazon medicinal plant Croton cajucara. This study aims to examine the potential effects of t-DCTN on hemodynamic parameters that include resting arterial blood pressure and heart rate in vivo, and on left atrial force, spontaneous beating atria, and aortic rings of rats in vitro. Intravenous bolus injections of t-DCTN (5, 10, or 15 mg/kg) to urethane anesthetized normotensive rats reduced the mean arterial pressure and heart rate in a dose-dependent manner. The hypotensive effect of t-DCTN (10 mg/kg) appears not mediated through effects on the muscarinic cholinergic receptor, beta-adrenoceptor, or ganglionic blockade, for it was not affected by atropine, propranolol, or hexamethonium but was abolished by N(w)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor. The diterpene t-DCTN showed no significant influence on inotropism. In isolated rat aortic rings with intact or denuded endothelium, t-DCTN relaxed the tonic contraction induced by phenylephrine (1 microM). Its vasorelaxant effect seen at smaller concentrations in endothelium intact preparations was, however, abolished in endothelium denuded or in l-NAME treated tissues. These data indicate the hypotensive and bradycardia effects of t-DCTN, possibly related in part to the release of nitric oxide and in part to direct effects on vascular smooth muscle, and cardiac pacemaker activity.