Development and validation of a stability-indicating HPLC method for the determination of buclizine hydrochloride in tablets and oral suspension and its application to dissolution studies

A method using liquid chromatography has been developed and validated for determination of buclizine in pharmaceutical formulations and in release studies. Isocratic chromatography was performed on a C18 column with methanol:water (80:20 v/v, pH 2.6) as mobile phase, at a flow rate of 1.0 mL/min, and UV detection at 230 nm. The method was linear, accurate, precise, sensible and robust. The dissolution test was optimized and validated in terms of dissolution medium, apparatus agitation and rotation speed. The presented analytical and dissolution procedures can be conveniently adopted in the quality and stability control of buclizine in tablets and oral suspension.

DI-HIDROCUCURBITACINA B: SEMI-SÍNTESE DE NOVOS DERIVADOS GLICOSILADOS

In the last ten years, the interest in natural and semi-synthetic cucurbitacin derivatives has increased, primarily due their cytotoxic and anti-tumoral activities. However, the isolation of glycosylated cucurbitacins has been difficult due the presence of β-glucosidase enzyme. With the aim of obtaining new glycosylated derivatives, the glycosylation of dihydrocucurbitacin B under Köenigs-Knorr and imidate reaction conditions was studied. Novel glycoside derivatives 16-(1,2-orthoacetate-3,4,6-tri-O-acetyl-α-D-glucopyranosyl)-dihydrocucurbitacin B (2), 2-O-β-D-2,3,4,6-tetra-O-acetyl-galactopyranosyl dihydrocucurbitacin B (3) and 2-O-β-D-galactopyranosyl dihydrocucurbitacin B (4) were synthesized for the first time in 17% (2 and 3) and 48% (4) yields.

DISTRIBUIÇÃO, ATIVIDADE BIOLÓGICA, SÍNTESE E MÉTODOS DE PURIFICAÇÃO DE PODOFILOTOXINA E SEUS DERIVADOS

Podophyllotoxin is the most studied lignan because of its use as an antimitotic agent and because it is a precursor of pharmacologically active derivatives. This review describes the anticancer activities of podophyllotoxin and the different processes that have been developed for its extraction and purification from Podophyllum spp. In addition, the synthesis routes of this compound and the development of three semi-synthetic procedures to obtain etoposide, teniposide, and Etopophos are detailed.

HS-SPME-GC-MS ANALYSIS OF VOLATILE AND SEMI-VOLATILE COMPOUNDS FROM DRIED LEAVES OF Mikania glomerata Sprengel

This paper reports on the identification of volatile and semi-volatile compounds and a comparison of the chromatographic profiles obtained by Headspace Solid-Phase Microextraction/Gas Chromatography with Mass Spectrometry detection (HS-SPME-GC-MS) of dried leaves of Mikania glomerata Sprengel (Asteraceae), also known as 'guaco.' Three different types of commercial SPME fibers were tested: polydimethylsiloxane (PDMS), polydimethylsiloxane/divinylbenzene (PDMS/DVB) and polyacrylate (PA). Fifty-nine compounds were fully identified by HS-SPME-HRGC-MS, including coumarin, a marker for the quality control of guaco-based phytomedicines; most of the other identified compounds were mono- and sesquiterpenes. PA fibers performed better in the analysis of coumarin, while PDMS-DVB proved to be the best choice for a general and non-selective analysis of volatile and semi-volatile guaco-based compounds. The SPME method is faster and requires a smaller sample than conventional hydrodistillation of essential oils, providing a general overview of the volatile and semi-volatile compounds of M. glomerata.

Use of semi-seletive media for detection of Sclerotinia sclerotiorum on bean and soybean seeds

This work was aimed at evaluating the possibility of using bromophenol blue as an indicator for detecting the presence of Sclerotinia sclerotiorum in the seeds of dry-beans (Phaseolus vulgaris) and soybean (Glycine max), through incubation of the seeds on an agar medium and "blotter" substrates. The seeds were artificially inoculated with four S. sclerotiorum isolates, plated on the agar medium, named Neon, and on modified Neon agar media all incubated at 14 and 20 ºC for seven days in the dark. Half of the seeds inoculated were surface desinfested prior to plating on the medium. The seeds showing change of colour in the medium, from blue to light yellow, as well as formation of typical mycelium and sclerotia in some cases, were considered to be infected or contaminated by S. sclerotiorum. The two incubation temperatures compared did not show significant (P<0.05) differences in detection level for most of the isolates tested on the different media. According to results obtained in this study, the Neon agar medium with incubation at 14 or 20 ºC has proved to be a reliable and quick method for the detection of S. sclerotiorum mycelium in naturally infected seeds of bean and soybean.

Hentriacontane: a leaf hydrocarbon from Syzygium jambos with stimulatory effects on the germination of urediniospores of Puccinia psidii

A crude Sohxlet extract from leaves of Syzygium jambos was sequentially fractionated using a silica gel flash column. A bioassay based on the numbers of urediniospores of Puccinia psidii that germinated in 2% water agar detected an active stimulant of germination when the fraction eluted with 100% n-hexane was used. The active fraction induced up to 88% increase in germination when added to a spore suspension in mineral oil. The active fraction was characterized as a hydrocarbon by ¹H nuclear magnetic resonance, 13C nuclear magnetic resonance, and infrared analysis. Gas chromatography-mass spectrometry analysis indicated that the fraction was a long-chain 436 MW hydrocarbon with corresponding to C31H64, namely hentriacontane. This is the first time such a compound proved to be involved with stimulation of fungal spore germination. These results may contribute to better understanding the infection process of rusts.

Distribuição espacial da meleira do mamoeiro em zonas de trópico úmido e trópico semi-árido

O vírus da meleira, transmitido por Bemisia tabaci, é um dos maiores problemas da cultura do mamoeiro (Carica papaya), sendo responsável por perdas de até 100% na produção. Com o objetivo de compreender melhor sua epidemiologia e gerar subsídios para estudos da influência de fatores culturais e bioecológicos na dinâmica da doença, avaliou-se a distribuição espacial de plantas afetadas pela meleira em zonas de Trópico Úmido e Trópico Semi-árido, em 15 plantios comerciais em Eunápolis-BA e Petrolina-PE, entre jan. 2000 e mar. 2001. As áreas foram mapeadas anotando-se a posição de cada planta e a presença ou ausência de sintomas. Foram consideradas doentes aquelas que apresentavam frutos com exsudação espontânea do látex, látex muito fluido, frutos borrados e/ou folhas do ápice com o bordo necrosado. Pelas análises de seqüências ordinárias, índice de dispersão, áreas isópatas e ajuste dos logaritmos das variâncias binomial e de acordo com a lei de Taylor, observaram-se maior agregação nas linhas de plantio que entre linhas e uma agregação de mediana a forte em sub-áreas (1,4 < ID < 3,1). Na maioria dos lotes, as áreas de maior incidência concentraram-se nas bordas dos talhões, o que indica que a migração de vetores assume um papel importante na disseminação da doença. Em alguns casos foi possível detectar a presença de focos isolados no interior dos lotes, o que sugere a formação de colônias dos vetores e transmissão planta a planta a partir de inóculo secundário. Não foram observadas diferenças significativas na distribuição espacial de plantas doentes entre as áreas das duas regiões.

A Semi-Selective agar medium to detect the presence of Xanthomonas axonopodis pv. malvacearum in naturally infected cotton seed

A semi-selective agar medium was developed for detection of Xanthomonas axonopodis pv. malvacearum (Xam) in cotton (Gossypium hirsutum) seed. The basic medium was peptone-sucrose-agar (PSA). Criteria for the semi-selective medium were the typical colony characters of Xam and its pathogenicity on cotton. Several systemic fungicides and antibiotics in different concentrations were tested alone or in combination with others. The final composition of the semi-selective agar medium was established after several attempts in order to inhibit most of the fungal and bacterial saprophytes and favour the development of Xam. It contained PSA + cyclohexamide, cephalexin, pencycuron, triadimenol and tolylfluanid. The bacteria were recovered from naturally infected seeds by the direct plating of 2,000 surface disinfected seeds on the semi-selective medium. The recovery of the pathogen from naturally infected leaf tissues and in dilution plating, on semi-selective medium and on nutrient agar, were comparable. Among the three detection methods tested, the semi-selective medium was found to be the most reliable and quantifiable. Degree of severity of angular leaf spot in the field was not always correlated with the level of infection in the seed. This is the first report of a semi-selective agar medium to detect the presence of Xam in naturally infected cotton seed.

Detecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijão em meio de cultura semi seletivo

Para a detecção de Xanthomonas axonopodis pv. phaseoli (Xap) em extratos de sementes inteiras e moídas de feijão, naturalmente contaminadas e sadias foram utilizados os meio de cultura semi-seletivos XCP1 e MT e o não seletivo 523 de Kado & Hesket. Os extratos de sementes foram também inoculados em folhas primárias de feijoeiro. O meio de cultura semi-seletivo XCP1 foi o mais eficiente na quantificação e detecção de Xap em extratos de sementes inteiras de feijão. Os extratos de sementes contaminadas, inoculados nas folhas primárias de feijoeiro produziram sintomas do crestamento bacteriano comum.

Analysis of the structural and electronic properties of 1-(5-Hydroxymethyl - 4 -[ 5 - (5-oxo-5-piperidin-1-yl-penta-1,3dienyl)-benzo[1,3]dioxol-2-yl] -tetrahydro-furan-2-yl)-5-methyl-1H-pyrimidine-2,4dione molecule

The structural and electronic properties of 1-(5-Hydroxymethyl - 4 -[ 5 - (5-oxo-5-piperidin- 1 -yl-penta- 1,3 -dienyl)-benzo [1,3] dioxol- 2 -yl]-tetrahydro -furan-2 -yl)-5-methy l-1Hpyrimidine-2,4dione (AHE) molecule have been investigated theoretically by performing density functional theory (DFT), and semi empirical molecular orbital calculations. The geometry of the molecule is optimized at the level of Austin Model 1 (AM1), and the electronic properties and relative energies of the molecules have been calculated by density functional theory in the ground state. The resultant dipole moment of the AHE molecule is about 2.6 and 2.3 Debyes by AM1 and DFT methods respectively, This property of AHE makes it an active molecule with its environment, that is AHE molecule may interacts with its environment strongly in solution.

Characterization of Citrus tristeza virus isolates from grapefruit (Citrus paradisi Macf.) accessions of Citrus Active Germplasm Bank

Citrus tristeza virus (CTV) isolates from 35 grapefruit accessions belonging to Citrus Active Germplasm Bank of the "Instituto Agronômico de Campinas" located at the "Centro APTA Citros Sylvio Moreira", Cordeirópolis, São Paulo state, Brazil, were characterized and evaluated through symptoms in the trees, biological indexing, immunological diagnosis with different monoclonal antibodies and SSCP analysis (single-strand conformation polymorphism) of the coat protein gene. Symptomatology indicated that, in general, the group of plants with smaller canopy volume and severe stem pitting differed significantly from the group that presented greater vegetative development and mild to moderate stem pitting. However, the isolates from most of the accessions induced mild reaction on Mexican lime. The serological evaluation through the DAS-ELISA using monoclonal antibodies did not reveal any association between virus titer in the plant tissue and symptoms. The reaction with different monoclonal antibodies and the distinct electrophoresis patterns obtained through SSCP showed that there is a high degree of diversity among the isolates that infect these grapefruit accessions. High complexity within the same isolate was also observed in the SSCP profiles. This finding indicates that the CTV isolates from these plants are a complex mixture of CTV haplotypes. Similar SSCP banding patterns were observed among some plants with strong stem pitting symptoms, and among some plants with weak or moderate stem pitting symptoms.

Meio de cultura semi-seletivo para detecção de Curtobacterium flaccumfaciens pv. flaccumfaciens em solo e sementes de feijoeiro

A murcha-de-curtobacterium, causada pela bactéria Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), é um importante problema para o cultivo de feijão (Phaseolus vulgaris L.) na região Centro-Sul do Brasil. A principal forma de disseminação da bactéria é por sementes contaminadas. Embora a ocorrência desta doença seja relativamente recente no Brasil, Cff tem sido disseminado rapidamente nas regiões produtoras de feijão do país. Este trabalho teve como objetivo ajustar o meio de cultura CNS (Corynebacterium Nebraskense Selective Medium) para recuperação e detecção de Cff em solo e sementes de feijoeiro. Suspensões provenientes da lavagem de amostras de solo e sementes contaminadas pela bactéria foram plaqueadas no meio de cultura CNS - modificado. Os ajustes realizados no meio de cultura possibilitaram a recuperação eficiente de Cff presente em solo e sementes contaminadas. As modificações estabelecidas para o meio CNS foram a redução da concentração de sulfato de polimixina para 16 mg/l, exclusão do componente ciclohexamida e substituição do fungicida Daconil 2787-F (530 mg/ml de chlorothalonil) pelo Dacostar 500 (500 mg/ml de chlorothalonil).

Meio semi-seletivo para detectar Xanthomonas axonopodis pv. phaseoli em sementes de feijoeiro e sua erradicação através do tratamento de sementes com o fungicida tolylfluanid

Estudou-se a eficiência do meio semi-seletivo desenvolvido para Xanthomonas axonopodis pv. malvacearum (Xam) do algodoeiro, com algumas modificações, na detecção de Xanthomonas axonopodis pv. phaseoli (Xap) em sementes de feijoeiro, bem como a eficiência do fungicida tolylfluanid na erradicação de Xap através do tratamento de sementes. Foram utilizadas sementes naturalmente infectadas por Xap, procedentes de diferentes municípios do Estado do Paraná. Houve o desenvolvimento de colônias bacterianas ao redor das sementes infectadas sob meio semi-seletivo entre seis e 12 dias de incubação. A freqüência de recuperação de colônias da bactéria em relação ao meio Agar nutriente (AN) variou entre 30 a 112%. O crescimento de Xap foi inibido a concentrações entre 54 a 1500 ppm do fungicida tolylfluanid. A bactéria foi recuperada das sementes não tratadas em meio semi-seletivo, mas não das sementes tratadas em solução do fungicida tolylfluanid (1,20g/L água). Em casa de vegetação, das 400 sementes tratadas com tolylfluanid não foi produzida nenhuma planta com sintomas da doença até 30 dias após a semeadura, enquanto que 9,75% das sementes não tratadas apresentaram sintomas de Xap. Em um outro experimento, das 1200 sementes não tratadas um total de 7,08% e 11,67% das plantas mostraram-se com sintomas da doença, enquanto que das sementes tratadas 0,5% e 2,4% apresentaram sintomas, 26 e 46 dias após a semeadura, respectivamente.

Distribution and role of macrophytes in coastal lagoons : implications of critical shifts

Crossroads, crucibles and refuges are three words that may describe natural coastal lagoon environments. The words refer to the complex mix of marine and terrestrial influences, prolonged dilution due to the semi-enclosed nature and the function of a habitat for highly diverse plant and animal communities, some of which are endangered. To attain a realistic picture of the present situation, high vulnerability to anthropogenic impact should be added to the description. As the sea floor in coastal lagoons is usually entirely photic, macrophyte primary production is accentuated compared with open sea environments. There is, however, a lack of proper knowledge on the importance of vegetation for the general functioning of coastal lagoon ecosystems. The aim of this thesis is to assess the role of macrophyte diversity, cover and species identity over temporal and spatial scales for lagoon functions, and to determine which steering factors primarily restrict the qualitative and quantitative composition of vegetation in coastal lagoons. The results are linked to patterns of related trophic levels and the indicative potential of vegetation for assessment of general conditions in coastal lagoons is evaluated. This thesis includes five field studies conducted in flads and glo-flads in the brackish water northern Baltic Sea. Flads and glo-flads are defined as a Baltic variety of coastal lagoons, which due to an inlet threshold and post-glacial landuplift slowly will be isolated from the open sea. This process shrinks inlet size, increases exposure and water retention, and is called habitat isolation. The studied coastal lagoons are situated in the archipelago areas of the eastern coast of Sweden, the Åland Islands and the south-west mainland of Finland, where land-uplift amounts to ca. 5 mm/ per year. Out of 400 evaluated sites, a total of 70 lagoons varying in inlet size, archipelago position and anthropogenic influence to cover for essential environmental variation were chosen for further inventory. Vegetation composition, cover and richness were measured together with several hydrographic and morphometric variables in the lagoons both seasonally and inter-annually to cover for general regional, local and temporal patterns influencing lagoon and vegetation development. On smaller species-level scale, the effects of macrophyte species identity and richness for the fish habitat function were studied by examining the influence of plant interaction on juvenile fish diversity. Thus, the active election of plant monoand polycultures by fish and the diversity of fish in the respective culture were examined and related to plant height and water depth. The lagoons and vegetation composition were found to experience a regime shift initiated by increased habitat isolation along with land-uplift. Vegetation composition altered, richness decreased and cover increased forming a less isolated and more isolated regime, named the vascular plant regime and charophyte regime, respectively according to the dominant vegetation. As total phosphorus in the water, turbidity and the impact of regional influences decreased in parallel, the dominance of charophytes and increasing cover seemed to buffer and stabilize conditions in the charophyte regime and indicated an increased functional role of vegetation for the lagoon ecosystem. The regime pattern was unaffected by geographical differences, while strong anthropogenic impact seemed to distort the pattern due to loss of especially Chara tomentosa L. in the charophyte regime. The regimes were further found unperturbed by short-time temporal fluctuations. In fact the seasonal and inter-annual dynamics reinforced the functional difference between the regimes by the increasing role of vegetation along habitat isolation and the resemblance to lake environments for the charophyte regime. For instance, greater total phosphorus and chlorophyll a concentrations in the water in the beginning of the season in the charophyte regime compared with the vascular plant regime presented a steeper reduction to even lower values than in the vascular plant regime along the season. Despite a regional importance and positive relationship of macrophyte diversity in relation to trophic diversity, species identity was underlined in the results of this thesis, especially with decreasing spatial scale. This result was supported partly by the increased role of charophytes in the functioning of the charophyte regime, but even more explicitly by the species-specific preference of juvenile fish for tall macrophyte monocultures. On a smaller species-level scale, tall plant species in monoculture seemed to be able to increase their length, indicating that negative selection forms preferred habitat structures, which increase fish diversity. This negative relationship between plant and fish diversity suggest a shift in diversity patterns among trohic levels on smaller scale. Thus, as diversity patterns seem complex and diverge among spatial scales, it might be ambiguous to extend the understanding of diversity relationships from one trophic level to the other. All together, the regime shift described here presents similarities to the regime development in marine lagoon environments and shallow lakes subjected to nutrient enrichment. However, due to nutrient buffering by vegetation with increased isolation and water retention as a consequence of the inlet threshold, the development seems opposite to the course along an eutrophication gradient described in marine lagoons lacking an inlet threshold, where the role of vegetation decreases. Thus, the results imply devastating consequences of inlet dredging (decreasing isolation) in terms of vegetation loss and nutrient release, and call for increased conservational supervision. Especially the red listed charophytes would suffer negatively from such interference and the consequences are likely to also deteriorate juvenile fish production. The fact that a new species to Finland, Chara connivens Salzm. Ex. Braun 1835 was discovered during this study further indicates a potential of the lagoons serving as refuges for rare species.

The Sottunga-Jurmo shear zone : structure and deformation history of a crustal-scale ductile shear zone in SW Finland

The aim of this study is to gain a better understanding of the structure and the deformation history of a NW-SE trending regional, crustal-scale shear structure in the Åland archipelago, SW Finland, called the Sottunga-Jurmo shear zone (SJSZ). Approaches involving e.g. structural geology, geochronology, geochemistry and metamorphic petrology were utilised in order to reconstruct the overall deformation history of the study area. The study therefore describes several features of the shear zone including structures, kinematics and lithologies within the study area, the ages of the different deformation phases (ductile to brittle) within the shear zone, as well as some geothermobarometric results. The results indicate that the SJSZ outlines a major crustal discontinuity between the extensively migmatized rocks NE of the shear zone and the unmigmatised, amphibolite facies rocks SW of the zone. The main SJSZ shows overall dextral lateral kinematics with a SW-side up vertical component and deformation partitioning into pure shear and simple shear dominated deformation styles that was intensified toward later stages of the deformation history. The deformation partitioning resulted in complex folding and refolding against the SW margin of the SJSZ, including conical and sheath folds, and in a formation of several minor strike-slip shear zones both parallel and conjugate to the main SJSZ in order to accommodate the regional transpressive stresses. Different deformation phases within the study area were dated by SIMS (zircon U-Pb), ID-TIMS (titanite U-Pb) and 40Ar/39Ar (pseudotachylyte wholerock) methods. The first deformation phase within the ca. 1.88 Ga rocks of the study area is dated at ca. 1.85 Ga, and the shear zone was reactivated twice within the ductile regime (at ca. 1.83 Ga and 1.79 Ga), during which the strain was successively increasingly partitioned into the main SJSZ and the minor shear zones. The age determinations suggest that the orogenic processes within the study area did not occur in a temporal continuum; instead, the metamorphic zircon rims and titanites show distinct, 10-20 Ma long breaks in deformation between phases of active deformation. The results of this study further imply slow cooling of the rocks through 600-700ºC so that at 1.79 Ga, 2 the temperature was still at least 600ºC. The highest recorded metamorphic pressures are 6.4-7.1 kbar. At the late stages or soon after the last ductile phase (ca. 1.79 Ga), relatively high-T mylonites and ultramylonites were formed, witnessing extreme deformation partitioning and high strain rates. After the rocks reached lower amphibolite facies to amphibolite-greenschist facies transitional conditions (ca. 500-550ºC), they cooled rapidly, probably due to crustal uplift and exhumation. The shear zone was reactivated at least once within the semi-brittle to brittle regime between ca. 1.79 Ga and 1.58 Ga, as evidenced by cataclasites and pseudotachylytes. In summary, the results of this study suggest that the Sottunga-Jurmo shear zone (and the South Finland shear zone) defines a major crustal discontinuity, and played a central role in accommodating the regional stresses during and after the Svecofennian orogeny.