Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As lectinas da subtribo Diocleinae pertencem à famÃlia de Leguminosas e sÃo caracterizadas pela alta homologia entre suas sequÃncias de aminoÃcidos. Apesar da alta similaridade estrutural o mesmo nÃo acontece nas atividades biolÃgicas das lectinas dessa famÃlia. Estudos mostram que a modificaÃÃo de poucos aminoÃcidos em suas sequÃncias à capaz de provocar grandes alteraÃÃes nas atividades biolÃgicas dessas lectinas, dessa forma o entendimento mais detalhado das estruturas tridimensionais dessas proteÃnas à essencial para a anÃlise da relaÃÃo entre estrutura e funÃÃo. A lectina de Dioclea violacea foi purificada por cromatografia de afinidade em coluna de Sephadex G-50. A proteÃna foi cristalizada na presenÃa do ligante X-Man e os cristais foram obtidos pelo mÃtodo de difusÃo de vapor em matriz esparsa por gota suspensa. Foram utilizando os kit âcristal screenâ I e II da Hampton Research e a condiÃÃo que obteve melhor cristal foi a condiÃÃo 33 do kit I composta por 4M Formato de sÃdio. O cristal foi difratado a 2,61à e apresenta grupo espacial I222 com cela unitÃria com dimensÃes de a = 61,34, b = 66,11, c = 106,69à e Ãngulos de α = β = γ = 90Â. Foi observada a presenÃa de um monÃmero na unidade assimÃtrica contendo 42,04% de solvente no cristal. A substituiÃÃo molecular foi feita utilizando a estrutura da lectina de Dioclea rostrata (PDB: 2ZBJ). O refinamento final obteve Rfactor de 0,23 e Rfree de 0,27 com ausÃncia aminoÃcidos em regiÃes nÃo permitidas do grÃfico de Ramachandran. O ligante X-Man foi co-cristalizado e sua estrutura foi observada perfeitamente encaixada no domÃnio de reconhecimento a carboidratos. Em relaÃÃo ao equilÃbrio dÃmero tetrÃmero, a lectina de Dioclea violacea apresenta o aminoÃcido HIS 131 e a posiÃÃo da HIS 51 à semelhante ao mesmo aminoÃcido da Dioclea grandiflora, caracterizando esta lectina como tetrÃmero mesmo em pH baixo. Foram feitas comparaÃÃes entre as atividades biolÃgicas de outras lectinas do gÃnero Dioclea e as distÃncias entre os resÃduos do sÃtio de ligaÃÃo a carboidrato. Essa analise mostrou que a variaÃÃo nessas distÃncias influi no efeito e eficÃcia da atividade vasorelaxante em aorta de ratos. / Lectins from subtribe Diocleinae belong to the family from Leguminosae and are characterized by high homology among their amino acid sequences. Despite this high structural similarity the same is not true in the biological activities from this lectin family. Studies show that the modification of a few amino acids can cause large changes in biological activities of these lectins. Thus more detailed understanding of three dimensional structures of these proteins is essential for structure/function analyses. The Dioclea violacea lectin was purified by affinity chromatography on a column of Sephadex G-50. The protein was crystallized in the presence of the ligand X-Man and the crystals were obtained by the vapor diffusion method in hanging drop by sparse matrix. Crystallization kits âCrystal Screen I and IIâ from Hampton Research were used to obtain protein crystals and the better condition was 33 from kit I4 M sodium formate. The crystal was diffracted to 2.61à with space group I222 with unit cell dimensions a = 61.34; b = 66.11; c = 106.69à and α = β = γ = 90Â. It was observed the presence of a monomer in asymmetric unit containing 42.04% of solvent in the crystal. The molecular replacement was made using Dioclea rostrata structure (PDB: 2ZBJ). The final refinement obtained Rfactorof 0.23 and Rfree of 0.27 in absence of any amino acid in regions not allowable in Ramachandran plot. The structure of X-Man was co-crystallized and observed perfectly placed in the carbohydrate recognition domain. Regarding the balance of the dimmer-tetramer associations of plant lectins, the lectin from Dioclea violacea has the amino acid HIS 131 and the position of HIS 51 is similar to the same amino acid in Dioclea grandiflora lectin, characterizing these lectins as a tetramer even at low pH. It was made comparisons between the differences in biological activities of other lectins from Diocleainae and the distances of the residues from carbohydrate site. It was observed that differences in biological activities in vasorelaxant effects on vascular smooth muscle are probably related to the distances between the residues that compose the carbohydrate domain.

The methylene chloride and acetone extracts of Formosan soft coral Clavularia violacea (Collected at Green Island) were found to exhibit significant cytotoxicity against P-388, A-549 and HT-29 cancer cell lines. Chromatographic separation let to the isolation of one prostanoid, 1, two cembranoid deterpenes, 2 and 3, one aromadedrane-type sesquiterpene, 4, one neodollabellane diterpene, 5, (+)-curcuphenol, 6 and two sesquiterpene lactones, 7 and 8. Compounds 1, 2, 4, and were new compounds. Compounds 1-5 exhibited significant cytotoxicity against P-388, HT-29 and A-549 cancer cell.

The present study was undertaken to determine the effects of varying light intensities on the morphology and physiology of the soft coral Pachyclavularia violacea. The soft corals P. violacea were treated by LED light and the illumination ranged from 50 to 200 £gmol photon m-2 s-1, i.e. high light (200 £gmol photon m-2 s-1), medium light (140 £gmol photon m-2 s-1), medium-low light (100 £gmol photon m-2 s-1 and low light (50 £gmol photon m-2 s-1). The theca length, the density of zooxanthellae and the concentrations of chlorophyll a and proteins were measured to evaluate the response of P. violacea to different light intensities. A significant longer theca length was found in the group of medium-low light (5¡V 9mm) than the groups of high and low light intensities (1-2mm) (p<0.001). And, the theca length in the groups of medium and medium-low was more close to their source population. Over the 6-month experimental periods, the densities of zooxanthellae in the groups of medium and medium-low were in the range of 1.5 ~3.8 x 105 (N/mg) which were significantly higher than other groups (p<0.01). The concentrations of chlorophyll a in the low light group were 0.5~ 2.0 (ng/mg) during the experimental periods which were significantly higher than other groups (p<0.05). In the fifth and sixth months, the concentrations of chlorophyll a per zooxanthellae in the group of medium-low were in the range of 0.4~5.8 Chl. a ¡Ñ 10-5 (ng/zoox.) which were significantly higher than other groups (p<0.01). The protein content in the group of low light was significantly lower than other groups in the fifth month. And, the protein contents were decreased significantly in all groups in the sixth month. Based on the results, it is concluded that theca length and the density of zooxanthellae were sensitive responses to light adaptation. The light intensity about 100 £gmol photon m-2 s-1 might be an appropriate range to culture the soft coral P. violacea because the theca length in the group was close to its source population.

BEZERRA, Maria Júlia Barbosa. Resolução da estrutura tridimensional da lectina de Dioclea violacea Mart para estudo da relação estrutura-função. 2011. 71 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2011. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-06-29T12:46:55Z No. of bitstreams: 1 2011_dis_mjbbezerra.pdf: 3567822 bytes, checksum: 02df5a11f781364615e5b2190b539688 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:12:28Z (GMT) No. of bitstreams: 1 2011_dis_mjbbezerra.pdf: 3567822 bytes, checksum: 02df5a11f781364615e5b2190b539688 (MD5) / Made available in DSpace on 2016-08-02T20:12:28Z (GMT). No. of bitstreams: 1 2011_dis_mjbbezerra.pdf: 3567822 bytes, checksum: 02df5a11f781364615e5b2190b539688 (MD5) Previous issue date: 2011 / Lectins from subtribe Diocleinae belong to the family from Leguminosae and are characterized by high homology among their amino acid sequences. Despite this high structural similarity the same is not true in the biological activities from this lectin family. Studies show that the modification of a few amino acids can cause large changes in biological activities of these lectins. Thus more detailed understanding of three dimensional structures of these proteins is essential for structure/function analyses. The Dioclea violacea lectin was purified by affinity chromatography on a column of Sephadex G-50. The protein was crystallized in the presence of the ligand X-Man and the crystals were obtained by the vapor diffusion method in hanging drop by sparse matrix. Crystallization kits “Crystal Screen I and II” from Hampton Research were used to obtain protein crystals and the better condition was 33 from kit I4 M sodium formate. The crystal was diffracted to 2.61Å with space group I222 with unit cell dimensions a = 61.34; b = 66.11; c = 106.69Å and α = β = γ = 90º. It was observed the presence of a monomer in asymmetric unit containing 42.04% of solvent in the crystal. The molecular replacement was made using Dioclea rostrata structure (PDB: 2ZBJ). The final refinement obtained Rfactorof 0.23 and Rfree of 0.27 in absence of any amino acid in regions not allowable in Ramachandran plot. The structure of X-Man was co-crystallized and observed perfectly placed in the carbohydrate recognition domain. Regarding the balance of the dimmer-tetramer associations of plant lectins, the lectin from Dioclea violacea has the amino acid HIS 131 and the position of HIS 51 is similar to the same amino acid in Dioclea grandiflora lectin, characterizing these lectins as a tetramer even at low pH. It was made comparisons between the differences in biological activities of other lectins from Diocleainae and the distances of the residues from carbohydrate site. It was observed that differences in biological activities in vasorelaxant effects on vascular smooth muscle are probably related to the distances between the residues that compose the carbohydrate domain. / As lectinas da subtribo Diocleinae pertencem à família de Leguminosas e são caracterizadas pela alta homologia entre suas sequências de aminoácidos. Apesar da alta similaridade estrutural o mesmo não acontece nas atividades biológicas das lectinas dessa família. Estudos mostram que a modificação de poucos aminoácidos em suas sequências é capaz de provocar grandes alterações nas atividades biológicas dessas lectinas, dessa forma o entendimento mais detalhado das estruturas tridimensionais dessas proteínas é essencial para a análise da relação entre estrutura e função. A lectina de Dioclea violacea foi purificada por cromatografia de afinidade em coluna de Sephadex G-50. A proteína foi cristalizada na presença do ligante X-Man e os cristais foram obtidos pelo método de difusão de vapor em matriz esparsa por gota suspensa. Foram utilizando os kit “cristal screen” I e II da Hampton Research e a condição que obteve melhor cristal foi a condição 33 do kit I composta por 4M Formato de sódio. O cristal foi difratado a 2,61Å e apresenta grupo espacial I222 com cela unitária com dimensões de a = 61,34, b = 66,11, c = 106,69Å e ângulos de α = β = γ = 90º. Foi observada a presença de um monômero na unidade assimétrica contendo 42,04% de solvente no cristal. A substituição molecular foi feita utilizando a estrutura da lectina de Dioclea rostrata (PDB: 2ZBJ). O refinamento final obteve Rfactor de 0,23 e Rfree de 0,27 com ausência aminoácidos em regiões não permitidas do gráfico de Ramachandran. O ligante X-Man foi co-cristalizado e sua estrutura foi observada perfeitamente encaixada no domínio de reconhecimento a carboidratos. Em relação ao equilíbrio dímero tetrâmero, a lectina de Dioclea violacea apresenta o aminoácido HIS 131 e a posição da HIS 51 é semelhante ao mesmo aminoácido da Dioclea grandiflora, caracterizando esta lectina como tetrâmero mesmo em pH baixo. Foram feitas comparações entre as atividades biológicas de outras lectinas do gênero Dioclea e as distâncias entre os resíduos do sítio de ligação a carboidrato. Essa analise mostrou que a variação nessas distâncias influi no efeito e eficácia da atividade vasorelaxante em aorta de ratos.

Les microalgues rouges de l’espèce Rhodella violacea produisent un exopolysaccharide soluble dans le milieu de culture. Au cours de ce travail, les conditions optimales de production ont été déterminées en étudiant l’activité photosynthétique et le milieu de culture des microalgues. La croissance cellulaire et la production d’EPS de R. violacea sont optimales avec un milieu de culture f/2 modifié, supplémenté en azote et en phosphore. L’irradiance optimale est de 420 μmol de photons.m-2.s-1, sous une température de 24°C avec un pH de 8,3. La culture de la microalgue en photobioréacteur de 5 L a permis la production optimale de 0,5 g.L-1 de polymère. Le polysaccharide produit est un protéoglycane de type xylane sulfaté et de haute masse molaire (1,2 106 g.mol-1), contenant également du rhamnose, du glucose, de l’arabinose, du galactose et de l’acide glucuronique. L’effet antiparasitaire des polymères de microalgues et de macroalgues a été testé sur des microsporidies, in vitro avec des fibroblastes de prépuce humain infestés par Encephalitozoon cuniculi et in vivo contre la nosémose des abeilles causée par Nosema ceranae. L’exopolymère de R. violacea empêche la croissance des microsporidies in vitro et in vivo, et conduit à une diminution de la mortalité des abeilles. D’autres molécules testées ont également une action antiparasitaire, les EPS de Porphyridium purpureum et marinum, et des carraghénanes sulfatés permettent également de diminuer la croissance des microsporidies et la mortalité des abeilles. / Red microalgae from Rhodella violacea species product a soluble exopolysaccharide release in the media. In this study, optimal culture conditions for exopolysaccharide production were investigated, following photosynthesis activity and culture conditions. This study allowed us to determinate R. violacea optimal media for growth and exopolysaccharide production, which is f/2 media supplemented in nitrogen and phosphorus. Optimal physicochemical parameters are an irradiance of 420 μmol photons.m-2.s-1, a temperature of 24°C, and a pH of 8.3. Photobioreactor of 5 L used to cultivate R. violacea in optimal conditions, gave 0.5 g.L-1 of EPS. Structural analysis of the EPS revealed the production of a proteoglycan, principally composed by xylose, sulfated and with a high molecular mass (1.2 106 g.mol-1). The polymer is complex, as it contains different monosaccharide: rhamnose, galactose, arabinose and glucuronic acid. The antiparasitic effect of polymers from microalgae, and macroalgae were investigated on microsporidia, in vitro against Encephalitozoon cuniculi using Human foreskin fibroblasts, and in vivo against Nosema ceranae using bees. Exopolysaccharide from R. violacea decreases microsporidia growth in vivo and in vitro. In addition, in vivo the polymer allows decrease in bees’ mortality. Polysaccharide from others origins also have antiparasitic effet, such as exopolymer from Porphyridium purpureum and marinum, and sulphated carragheenans which reduce microsporidia growth, and decrease bees’ mortality.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Lectinas de origem vegetal inibem quimiotaxia neutrofÃlica, provavelmente por aÃÃo competitiva com selectinas endÃgenas por um sÃtio glicosÃdico na membrana de cÃlulas endoteliais, leucÃcitos ou em componentes da matrix extracelular. Exploramos os efeitos de lectina isolada de sementes de Dioclea violacea (Dviol) na artrite induzida por zymosan (Azy). Ratos Wistar (180 a 220g) receberam injeÃÃo intra-articular (i.a.) de zymosan (Zy) (1mg) no joelho direito. A hiperalgesia foi avaliada pelo teste de incapacitaÃÃo articular, medido pelo tempo de suspensÃo da pata (TSP) em s/min. O influxo celular (IC) foi medido no lavado articular, obtido 6 h apÃs a injeÃÃo do Zy, por lavagem e aspiraÃÃo da articulaÃÃo. Grupos foram prÃ-tratados (30 min) antes do Zy com Dviol (1 - 30 Âg; i.a. ou 1 - 10mg/kg; e.v.), sendo comparados ao grupo nÃo-tratado (NT), que recebeu apenas Zy e o veÃculo. Outros grupos receberam apenas Dviol (0,3- 30 Âg) i.a. e o grupo Naive, apenas salina i.a. A administraÃÃo de Dviol (6mg/kg; e.v.), reduziu significantemente (p<0,01) a hiperalgesia (TSP= 14,08  1,4) quando comparado ao NT (TSP= 36,06  3,3). A injeÃÃo endovenosa de Dviol inibiu o IC (18.266,7  1.890,1; 14.633,3  3.207,2; 2.790  503,3 e 120  37,4 cÃlulas/mm3 para 1, 3, 6 e 10mg de Dviol, respectivamente) quando comparados ao NT (37.583,3  6.007,2 cÃlulas/mm3). A administraÃÃo i.a. de apenas Dviol (30 Âg) aumentou significativamente o TSP (15,5  0,9), em relaÃÃo ao NV. Dviol i.a. isolada (0,3 a 30; Âg), aumentou significantemente o IC (3.600  676; 4.958,3  1037,2 e 8.350  1.511,5 cÃlulas/mm3 para lectina 0,3; 3 e 30, respectivamente), comparado ao NV (858,3  389,5 cÃlulas/mm3). Dviol (10 Âg; i.a.), 30 min antes da injeÃÃo do Zy, inibiu significantemente a hiperalgesia (TSP = 20,15  2,2) (p<0,01), em relaÃÃo ao NT (TSP= 35,9  2,7). Dviol i.a. (1, 10 e 30&#956;g) reduziu significantemente o IC (18.266,7  1.890,1; 11.366,7  2.883,7 e 19.866,7  1.783,4 cÃlulas/mm3, respectivamente), comparado ao NT. A administraÃÃo e.v. de Dviol (6 mg/kg) + manose (0,1M), reverteu a inibiÃÃo da Dviol na hiperalgesia (TSP= 38,4  4,4) (P<0,01) e no IC (15200  1829,7 cÃlulas/mm3) da AZy, em relaÃÃo a Dviol (6mg/kg;e.v.) (TSP= 14,08  1,4 e IC= 2.790  503,3 cÃlulas/mm3). Os resultados demonstram, de maneira inÃdita, que a lectina de semente de Dviol tem aÃÃo antiinflamatÃria em artrite experimental. Esse efeito parece ser prioritariamente associado à ligaÃÃo da Dviol a sÃtios de polissacarÃdeos intra-articulares, embora nÃo possamos descartar que a ligaÃÃo a domÃnios protÃicos possa estar envolvida nessa aÃÃo / Plant-derived lectins inhibit neutrophil chemotaxis, probably through a competitive mechanism with endogenous selectins for a glycosidic residue in the membrane of endothelial cells, leukocytes or in components of the extracellular matrix. We investigated the effects of a lectin isolated from Dioclea violacea (Dviol) seeds in the zymosan-induced arthritis (Azy). Wistar rats (180 a 220g) received an intraarticular (i.a.) injection of 1 mg zymosan (Zy) into the right knee. The hyperalgesia was evaluated with the test for articular incapacitation, measured as the paw elevation time (PET) in s/1min. Cell influx (IC) was assessed in the joint exudate, obtained 6 h after injection of the Zy, through washing and aspiration of the joint. Groups were pretreated 30min before the Zy with Dviol (1 - 30 Âg; i.a. ou 1 - 10mg/kg; e.v.), and were compared to non-treated groups (NT), that received the Zy and the vehicle. Others groups received only Dviol (0,3 - 30Âg; i.a. and the naÃve animals (NV) received just saline i.a. The i.v. administration of Dviol (6mg/kg) significantly reduced the hyperalgesia (p<0.01) (PET= 14,08  1,4) as compared to NT (PET= 36,06  3,3). The i.v. injection of Dviol inhibited IC (18.266,7  1.890,1; 14.633,3  3.207,2; 2.790  503,3 e 120  37,4 cells/mm3 for the 1, 3, 6, and 10mg doses of Dviol, respectively). Dviol given i.a. isolated (30Âg) increased PET (15,5  0,9) significantly, as compared to NV. Dviol i.a. isolated (0,3 - 30 Âg) significantly increased IC (3.600  676; 4.958,3  1037,2 e 8.350  1.511,5 cells/mm3 for 0,3, 3 e 30 Âg, respectively), as compared to NV (858,3  389,5 cells/mm3). Dviol (10 Âg i.a.) given 30min before the Zy significantly inhibited the hyperalgesia (PET = 20,15  2,2) (p<0.01), as compared to NT (TSP= 9,9 Â1,2). Dviol i.a. (1, 10, and 30&#956;g) significantly reduced IC (18.266,7  1.890,1; 11.366,7  2.883,7 e 19.866,7  1.783,4 cells/mm3, respectively), as compared to NT. The i.v. administration of Dviol (6 mg/kg) + manose reversed the inhibitory action of Dviol in the hyperalgesia (PET= 38,4  4,4) (P<0.01) and in the IC (15200  1829,7 cells/mm3) of the AZy, as compared to Dviol (6mg/kg; i.v.) (TEP= 14,08  1,4 and IC= 2.790  503,3 cells/mm3). The results demonstrate, for the first time, that the lectin isolated from Dviol has anti-inflammatory effect in an experimental arthritis model. This effect appears to be prominently due to the coupling of Dviol to intrarticular polysaccharide residues. However, we can not exclude that coupling to protein residues may be also involved in this mechanism

The coral-inhabiting snail Coralliophila violacea is a common species in the Indo-Pacific coral reefs and it usually lives on the surface of its host, Porites spp. In this study, field survey on the distribution of C. violacea on poritid corals, Porites spp. among sites were conducted. And the influence of flow rate on the distribution of snails was also examined through a simulation model. The sampling sites were northeastern of Taiwan, Taitung, Penghu, Lutao, Hsiao-liu-chiu and Lanyu. The presence of spatial variability in the relative abundance of C. violacea on porited corals has been observed. The highest percentage of corals with snails was in northeastern Taiwan, i.e. 45% and the lowest ones were in Lutao and Lanyu, i.e. 12%. The distribution of snails among sites were heterogeneous (X2-test of independence; p<0.001). Significant differences of shell length in snails among sites were also found. Among them, the distribution pattern of shell length in snails from Lutao and Lanyu was not normal. Difference in the age-distribution of snails among sites was present. In general, five and six years old snails were most abundant. A lack of snails under two years old had been found in Lutao, Lanyu and Penghu. Based on the simulated downward trajectory of snail larvae, it was indicated that a minimum substratum for larval settlement varied with flow rates and depth of water.

The organic extracts of two marine soft corals Pachyclavularia violacea and Subergorgia suberosa, collected along the coast of Kenting, Taiwan, were found to exhibit significant cytotoxicities toward several cancer cell lines (Table 1). In order to discover bioactive compounds, we have investigated the chemical constituents of these two marine organisms. Investigation on P. violacea has led to the isolation of twenty-five compounds, including twenty-one new compounds, pachyclavulariolide G (1), pachyclavulariolide H (3), pachyclavulariolide I (4), pachyclavulariolides J¡VS (6¡V15), pachyclavulariaenones A¡VG (16¡V22), secopachyclavulariaenone A (23), and four known compounds pachyclavulariolide (2), pachyclavulariolide E (5), pachyclavulariolide A (24) and pachyclavulariolide B (25). Also, we have investigated the chemical constituents of S. suberosa. This study led to the isolation of fifteen compounds, including six new compounds, 2b-acetoxysubergorgic acid (27), subergorgiol (31), suberosols A¡VD (34¡V37), and nine known compounds, subergorgic acid (26), 2b-hydroxysubergorgic acid (28), methyl ester of subergorgic acid (29), 2b-acetoxy methyl ester of subergorgic acid (30), buddledin D (32), buddledin C (33), 5b-pregnan-3,20-dione (38), £G1- 5b-pregnen-3,20-dione (39) and 3a-acetoxy -5b-pregnan-20-one (40). Compounds 39, 40 were isolated from natural sources for the first time. Structures of these compounds were determined on the basis of chemical method and spectroscopic evidences. Cytotoxicities of these compounds against P-388, KB, A-549 and HT-29 cancer cell lines also were described. Compound, 7, 32, 33, 36, 37 have been found to show moderate activity toward the above cancer cell lines.

This dissertation mainly presented the investigation of natural products from two different Formosan gongonian soft corals, Briareum violacea and Junceella fragilis. Their extracts were examined by intensive chromatographic methods. Eighty-four compounds including fifty new briaranes were isolated, and parts of their biological activities were studied. Natural products investigation of the Taiwanese gorgonian octocoral Junceella fragilis found fifteen new briarane-type diterpenes, namely frajunolides E-S (1-15), ninteen known briaranes, eg. junceellin, junceellolides A-E¡Bjunceellolide K, 11£\,20£\-epoxy-4-deacetoxyjuncellolide D, umbraculolide A, junceellonoid A, juncin P, juncins Y-ZI, frajunolides A-D and praelolide, and a sterol, ergosterol peroxide. Soft coral B. violacea (Quoy and Gaimard) of Taiwan has isolated thirty-five new briarane-type diterpenoids, briaviolides A-Z (16-41) and viobrianolides A-I (42-50), in addition to fourteen known diterpene lactones, stylatulide lactone, excavatolide A, 9-deacetylstylatulide lactone, 4£]-acetoxy-9-deacetylstylatulide lactone¡B Brianthein Z¡B, renillafoulin A, braexcavatolide E, braexcavatolide I, minabein-4, minabein-6, milolide K, solenolide A, and solenolides D-E. Structures of all above compounds were examined by physical and spectroscopic analyses including mass, IR, UV spectra, optical rotation and 1D, 2D NMR, as well as in comparison with the published data. Moreover, the structures of compounds 16, 32 and prarlolide were further confirmed by single X-ray crystallographic analysis. The stereochemistry of these compounds was investigated by NOESY method, and the configuration of compounds 32-38 were determined by Circular Dichroism spectroscopic analysis. Cytotoixcity and in vitro anti-inflammatory activities were studied by Dr. Kuo, Yao-Haur and Dr. Hwang, Tsong-Long, respectively. Junceellin, junceellolide B and juncin ZI exhibited weak cytotoxicity against Hep2 (Human laryngeal carcinoma), Doay (Human medulloblastoma), WiDr (Human colon adenocarcinoma), Hela (Human cervical epitheloid carcinoma). The in vitro anti-inflammatory activities of 1-7 were evaluated for inhibition of elastase release and for the generation of superoxide anion, as tested on human neutrophils. Compounds 1 and 6 exhibited weak inhibition of elastase release and superoxide anion at 10 £gg/mL. The biological activity analysis of compounds 16-50 are in progress.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Even though Tulbaghia violacea has been used to treat and manage epilepsy in South Africa by traditional medicine practitioners, no evidence in any literature has shown any scientific scrutiny of the effectiveness of the plant species in therapy. This project was intended, therefore, to investigate the anticonvulsant effect of the leaf methanol extract of Tulbaghia violacea by studying its effect against tonic convulsion induced by either pentylenetetrazole (PTZ), bicuculline, picrotoxin, strychnine or N-methyl-DL-aspartic acid (NMDLA) in mice. Qualitative phytochemical analysis, acute toxicity and HPLC studies were also carried out on the plant species. The doses of 200 (mg/kg, i.p.) and 400 (mg/kg, i.p.) of the leaf methanol extract of T. Violacae significantly delayed the onset of PTZ (100 mg/kg, i.p.)-induced tonic convulsion in a dose dependent manner. Leaf methanol extract of the plant species (200 mg/kg, i.p.) did not affect the incidence of PTZ (100 mg/kg, i.p.)-induced tonic convulsion while 400 mg/kg (i.p.) protected only one mouse against the tonic convulsion. Leaf methanol extract of Tulbaghia violacea (100mg/kg, i.p.) did not significantly affect the onset or incidence of PTZ (100 mg/kg, i.p.)- induce tonic convulsion. Phenobarbitone (12 mg/kg, i.p.), diazepam (0.5 mg/kg, i.p.) and muscimol (2mg/kg, i.p.) significantly delayed the onset of PTZ (100 mg/kg, i.p.)-induced tonic convulsion and also significantly reduced the number of animals convulsing. Muscimol (0.2 mg/kg, i.p.) did not significantly affect the onset or incidence of PTZ (100 mg/kg, i.p.)-induced tonic convulsion. However, combined therapy of sub effective doses of the leaf methanol extract of T. Violacea (100 mg/kg, i.p.) and muscimol (0.2 mg/kg, i.p.) significantly delayed the onset of PTZ (100mg/kg, i.p.)-induced tonic convulsion and but did not significantly reduce the number of animals convulsing. The combined therapy of sub effective doses of the leaf methanol extract of T. violacea (100 mg/kg, i.p.) and muscimol (0.2 mg/kg, i.p.) protected two of the mice against the tonic convulsion. Leaf methanol extract of Tulbaghia violacea (100-400 mg/kg, i.p.) significantly and dose dependently delayed the onset of tonic convulsion produced by bicuculline (30 mg/kg, i.p.), picrotoxin (20 mg/kg, i.p.) or NMDLA (400 mg/kg, i.p.)-induced tonic convulsion but did not affect the incidence of any of the convulsions. Phenobarbitone (12 mg/kg, i.p.), diazepam (0.5 mg/kg, i.p.) or muscimol (2 mg/kg, i.p.) significantly delayed the onset of bicuculline (30 mg/kg, i.p.) or picrotoxin (20 mg/kg, i.p.)-induced tonic convulsion and also significantly reduced the number of animals convulsing. Phenobarbitone (12 mg/kg, i.p.) or diazepam (0.5 mg/kg, i.p.) did affect significantly affect the onset or incidence of NMDLA (400 mg/kg, i.p.)-induced tonic convulsion. LY233053 (5 mg/kg, i.p.) significantly delayed the onset of tonic convulsion produced by NMDLA (400 mg/kg, i.p.) and also significantly reduced the number of animals convulsing. Leaf methanol extract of Tulbaghia violacea (200 and 400 mg/kg, i.p.) significantly delayed the onset of strychnine (2 mg/kg, i.p.)-induced tonic convulsion but did not significantly affect the number of mice convulsing. The dose of 100 mg/kg (i.p.) of leaf methanol extract of T. violacea did not significantly affect the onset or incidence of strychnine (2 mg/kg, i.p.)-induced tonic convulsion. Phenobarbitone (12 mg/kg, i.p.) significantly delayed the onset of strychnine (2 mg/kg, i.p.)-induced tonic convulsion and also significantly reduced the number of animals convulsing. Diazepam (0.5 mg/kg, i.p.) did not significantly delay the onset of strychnine (2 mg/kg, i.p.)-induced tonic convulsion and also did not significantly affect the number of mice convulsing. Phenytoin (30 mg/kg, i.p.) or DMSO (0.25 ml, i.p.) did not significantly affect the onset or incidence of bicuculline (30 mg/kg, i.p.), picrotoxin, strychnine or NMDLA-induced tonic convulsion. The qualitative phytochemical analysis of the plant species showed the presence of alkaloids, saponins, cardiac glycosides, flavonoids, triterpene steroids, quinones and tannins. The LD50 value obtained following oral administration of the leaf methanol extract of Tulbaghia violacea may be greater than 4000 mg/kg. The HPLC fingerprint of the leaf methanol extract of Tulbaghia violacea showed distinct peaks at the following retention times, 2.911, 3.269, 4.010, 7.597, and 15.122 min. The results obtained in this study indicate that the leaf methanol extract of Tulbaghia violacea has anticonvulsant activity. The results obtained also indicate that GABA, glutamic acid and glycine mechanisms may probably be involved in the anticonvulsant activity of the plant extract. The relatively high LD50 obtained for the plant species, given orally, indicate that it is safe in mice.