Conselho Nacional de Desenvolvimento Científico e Tecnológico / This work describes the chemical study of renewable natural resources of the State of Sergipe, Brazil, in that we analyzed the leaves and branches of mangabeira, Hancornia speciosa Gomes (Apocynaceae).This work is divided into 3 chapters. In the first part of the research was investigated the volatile chemical composition from leaves varying the drying time and harvest time. Analysis by GC/MS and GC/FID showed that there was a significant increase in the content of some oxygenated monoterpenes according to the drying of leaves, such as -terpineol (0,08±0,20 - 3,89±1,78 in fresh leaves to 0,93±0,03 - 5,21±0,65 in dried leaves), linalool (2,95±1,45 - 12,46±2,24 in fresh leaves to 2,78±1,08 - 17,16±2,83 in dried leaves), geraniol (1,04±0,34 - 12,80±1,23 in fresh leaves to 25,67±6,45- 46,56±4,67 in dried leaves), as well and a decrease in the relative percentage of alcohols such as (Z)-3-hexenol (46,81±3,27 - 78,45±7,75 in fresh leaves to 10,70±4,87 - 19,15±4,28 in dried leaves) and 1-hexanol (1,65±0,33 - 10,15±0,80 in fresh leaves to 0,07±0,05 - 4,59±1,55 in dried leaves).In the months with higher rainfall and temperature we observed a higher yield of essential oils from leaves of H. speciosa. In the second part of the work, the phytochemical study of the leaves and branches of this species allowed the isolation of 3 mixtures. Those containing a mixture of triterpenes -amyrin, -amirin and lupeol were isolated from the hexane partition of methanolic extract of the leaves, while a mixture of 9 3--O-acyl lupeol esters and another mixture of n-alkanes were isolated from hexane extract of the branches.Structure determination of the isolated compounds was based on analysis of spectroscopic methods. In the third part, we evaluated the antioxidant activity of essential oils from fresh and dried leaves, and the methanol extract of leaves and branches and their partitioned extracts, which was seen to goodability to scavenge free radicals butanol partition of the mathanol extract of leaves. We also investigate the cytotoxic activity of hexane and methanol extracts of leaves and branches against cancer cell lines MDA-MB-435 (melanoma) and SF-295 (brain) and HCT-8 (human colon carcinoma), which was not observed an inhibitory effect. / O presente trabalho descreve o estudo químico dos recursos naturais renováveis do estado de Sergipe, onde foram analisadas as folhas e ramos da mangabeira, Hancornia speciosa Gomes (Apocynaceae). Na primeira parte do trabalho foi investigada a composição química dos voláteis das folhas variando-se o tempo de secagem e a época de colheita. As análises através de CG/EM e CG/DIC permitiram verificar que, com a secagem das folhas, houve um aumento significativo nas percentagens relativas de alguns monoterpenos oxigenados como -terpineol (0,08±0,20 - 3,89±1,78 nas folhas frescas para 0,93±0,03 - 5,21±0,65 nas folhas secas), linalol (2,95±1,45 - 12,46±2,24 nas folhas frescas para 2,78±1,08 - 17,16±2,83 nas folhas secas), geraniol (1,04±0,34 - 12,80±1,23 nas folhas frescas para 25,67±6,45- 46,56±4,67 nas folhas secas), assim como um decréscimo nas percentagens de alcoóis como (Z)-3-hexenol (46,81±3,27 - 78,45±7,75 nas folhas frescas para 10,70±4,87 - 19,15±4,28 nas folhas secas) e 1- hexanol (1,65±0,33 - 10,15±0,80 nas folhas frescas para 0,07±0,05 - 4,59±1,55 nas folhas secas). Nos meses com valores de índice de pluviometria e temperatura elevados observou-se um maior rendimento de óleo essencial de H. speciosa. Na segunda parte do trabalho, o estudo fitoquímico das folhas e dos ramos desta espécie permitiu a identificação de 3 misturas. Destas uma mistura de triterpenos contendo -amirina, - amirina e lupeol foi identificada da partição hexânica do extrato metanólico das folhas, enquanto uma mistura contendo 9 ésteres 3--O-acil lupeol e uma outra mistura contendo 12 n-alcanos foram identificadas do extrato hexânico dos ramos. A determinação estrutural das substâncias foi baseada na análise dos dados espectroscópicos obtidos. Na terceira parte do trabalho, foram realizados testes de atividade antioxidante com os óleos essenciais das folhas frescas e secas, e com o extrato metanólico das folhas e dos ramos e suas respectivas partições, onde foi observada a boa capacidade de sequestrar radicais livres da partição butanólica do extrato metanólico das folhas. Além da investigação da citotoxicidade dos extratos hexânicos e metanólicos das folhas e dos ramos frente às linhagens de células tumorais MDA-MB435 (mama - humano) e SF-295 (glioblastoma - humano) e HCT-8 (colon), onde não foi observado um efeito inibitório.

L’étude chimique de 11 plantes du Gabon, dont certaines utilisées en médecine traditionnelle, a été réalisée à la recherche de composés antiplasmodiaux de structures nouvelles. Deux Apocynaceae (Pleiocarpa mutica Benth., Callichilia inaequalis Stapf) et une Gelsemiaceae (Mostuea brunonis Didr.) ont été plus spécifiquement étudiées, via une stratégie de déréplication fondée sur des réseaux moléculaires générés à partir de données CLHP-MS/MS (Molecular Networking) et annotés avec une base de données d’alcaloïdes indolomonoterpéniques mise au point au laboratoire (MIADB). Cette investigation a guidé un travail de fractionnement et d’isolement, qui a permis l’obtention d’alcaloïdes indolomonoterpéniques très originaux, en termes de décorations ou de modes d’assemblage.Des écorces de tiges de P. mutica, 7 dimères indolomonoterpéniques non décrits dans la littérature ont été obtenus : 5 sont des bis-aspidofractanes dont 4 sont reliés par un pont méthylène (pléiokomenines A-D) ; deux sont des dimères du type aspidofractane-éburnamine, analogues de la pléiomutine.Les tiges et les racines de C. inaequalis ont livré 2 bis-indoles nouveaux, analogues de la criophylline, dont le premier porteur d’un sulfate dans cette classe d’alcaloïdes, ainsi que l’inaequalisine A, premier indolomonoterpène monomérique lié à un reste phénylpropène via une liaison C-C.L’étude des tiges et des feuilles de M. brunonis a conduit à l’isolement de quatre nouveaux composés : un monomère de type sarpagine (16-epi-méthylester-panarine) et 3 dimères bis-vobasines inédits à pont sulfide (théionbrunonines A-C). Des molécules connues, mais pas toujours identifiées dans les genres étudiés, ont également été isolées. Plusieurs des composés nouveaux présentent une activité antiplasmodiale de l’ordre du µM in vitro sur une souche de Plasmodium falciparum chloroquino-résistante. / The chemical study of 11 Gabonese plant species, some being used in traditional medicine, was performed in search of antiplasmodial compounds with new structures. Two Apocynaceae (Pleiocarpa mutica Benth., Callichilia inaequalis Stapf) and a Gelsemiaceae (Mostuea brunonis Didr.) were more specifically investigated, using LC-MS/MS data in a dereplicative approach based on the “molecular networking” strategy, with an annotation performed using an “in-house” monoterpene indole alkaloids database (MIADB). This approach was used to guide the isolation of original alkaloids, in terms of substitution patterns or of linkage. From the stem bark of P. mutica, 7 previously undescribed bis-indoles were obtained: five are bis-aspidofractanes, four of them being linked by a methylene bridge (pleiokomenines A-D); Two are aspidofractane-eburnane dimers analogous to pleiomutine. The twigs and roots of C. inaequalis yielded 2 new bis-indoles analogous to criophylline, among which one is the first natural sulfate-bearing indole monoterpene. Inaequalisine A, the first monomeric indole monoterpene linked to a phenylpropene moiety by a C-C linkage was also obtained.The study of the twigs and leaves of M. brunonis lead to 4 new compounds: A monomeric sarpagine (16-epi-methylester-panarine) and 3 unknown bis-vobasines which constituting monomers are linked by a sulfide bridge (theionbrunonines A-C).Known compounds were also isolated, some of which were previously undescribed in the genera studied here. Several of the new molecules exhibited antiplasmodial in vitro activity in the micromolar range against a chloroquine-resistant strain of P. falciparum.

Landolphia kirkii is scrambling shrub forming an integral part of the flora along the coastal areas of north-eastern South Africa. The non-sustainable harvesting of fruit as food source, by monkeys and rural communities and the highly recalcitrant nature of their seeds threatens the continuation of the species. In addition, the ability of the plants to produce high quality rubber makes its long-term conservation highly desirable. Previously, attempts have been made to cryopreserve germplasm of L. kirkii, but no survival had been recorded at cryogenic temperatures of below -140ºC. The present study reports on the effects of rapid dehydration, chemical cryoprotectants and various cooling rates, thawing and imbibition treatments on survival of embryonic axes excised with cotyledons completely removed, as well as with 3 mm portion of each cotyledon attached, from fresh, mature, recalcitrant seeds of L. kirkii. Survival was assessed by the ability for both root and shoot development in in vitro culture, the tetrazolium test and electrolyte leakage readings. At seed shedding, embryonic axes were at the high mean water content of 2.24 g gˉ¹ (dry mass basis). All axes (with and without attached cotyledonary segments) withstood rapid (flash) drying to a water content of c. 0.28 g gˉ¹; however, the use of chemical cryoprotectants, singly or in combination, before flash-drying was lethal. Rapid cooling rates were detrimental to axes flash-dried to 0.28 g gˉ¹, with no explants showing shoot production after exposure to -196ºC and -210ºC. Ultrastructural examination revealed that decompartmentation and loss of cellular integrity were associated with viability loss after rapid cooling to cryogenic temperatures, although lipid bodies retained their morphology regardless of the thawing temperature employed. Furthermore, analysis of the lipid composition within embryos of L. kirkii revealed negligible amounts of capric and lauric acids, suggested to be the medium-chained saturated fatty acids responsible for triacylglycerol crystallisation when lipid-rich seeds are subjected to cryogenic temperatures. Hence, lipid crystallisation was not implicated in cell death following dehydration, exposure to cryogenic temperatures and subsequent thawing and rehydration. Rapid rehydration of embryonic axes of L. kirkii by direct immersion in a calcium-magnesium solution at 25ºC for 30 min (as apposed to slow rehydration on moistened filter paper or with rehydration in water) was associated with highest survival post-dehydration. Cooling at 1ºC minˉ¹ and 2ºC minˉ¹ facilitated survival of 70 and 75% respectively of axes with attached cotyledonary segments at 0.28 g gˉ¹ after exposure to - 70ºC. Viability retention of 40 and 45% were recorded when embryonic axes with attached cotyledonary segments were cooled at 14 and 15ºC minˉ¹ to temperatures below -180ºC. However, no axes excised without attached cotyledonary segments produced shoots after cryogenic exposure. The use of slow cooling rates is promising for cryopreservation of mature axes of L. kirkii, but only when excised with a portion of each cotyledon left attached. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Bouz L.: Biological activity of secondary plants metabolites V. Alkaloids of Vinca minor L. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2016. Summary extract obtained from dried aerial parts of Vinca minor L. was separated by column chromatography with petrol, chloroform and ethanol to 531 fractions. By further separation of fractions, following preparative thin layer chromatography and crystallization 2 alkaloidal compounds marked LB-2 and LB-3 were isolated. These compounds were identified by GC/MS, 1 H- and 13 C-NMR spectroscopy and by use of physical-chemical methods. The structure of compounds were elucidated as indole alkaloids (+)-vincaminoreine (LB-2) and (+)-vincamine (LB-3). Both substances were tested for their inhibition activity against human cholinesterases. (+)-Vincamine didn't exhibited in comparison with standard drugs (galanthamine IC50 AChE: 1,710 ± 0,065 µM, IC50 BChE: 42,30 ± 1,30 µM; huperzine A IC50 AChE: 0,033 ± 0,001 µM) any inhibition activity. On the other hand (+)-vincaminoreine exhibited fairly strong selective inhibition of BChE (IC50 = 8,71 ± 0,49 µM) with no inhibition of AChE. Key words: Vinca minor L., indole alkaloids, vincamine, vincaminoreine, Alzheimer disease,...

Vítovcová, A.: Biological activity of secondary plants metabolites IV. Alkaloids of Vinca minor L. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2016. Extract of Vinca minor L. was separated to fractions in column chromatography. Flash chromatography, preparative TLC and crystalisation led to isolation of two alkaloids from fraction number two and number five. Alkaloids were identified by GC/MS as (+)-vincaminorine and vincorine. These alkaloids were tested for their inhibition activity towards cholinesterase (AChE and BuChe). Obtained activities IC50 were compared with standards (galanthamine, huperzine A). The most interesting activity against galanthamine (AChE 1,710 ± 0,065 µM, BuChE 42,30 ± 1,30 µM) and huperzine A (AChE 0,033 ± 0,001 µM, BuChE > 1000 µM) showed vincorine (AChE > 1000 µM, BuChE 9,75 ± 0,45 µM). His activity toward BuChE is higher than the activity of both standards. Inhibition activities of (+)-vincaminorine (AChE 746,5 ± 84,13 µM, BuChE 684,32 ± 70,66 µM) are negligible. Key words: Alzheimerʼs disease, cholinesterase, Apocynaceae, Vinca minor L., indole alkaloids

1 ABSTRACT Kučerová J.: Biological activity of secondary plants metabolites VI. Alkaloids of Vinca minor L. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of pharmaceutical botany and ecology, Hradec Králové 2016, 67 pages. Isolation of alkaloids was done from extract of Vinca minor, family Apocynaceae. Individual substances obtained were examined for their ability to inhibit human acetylcholinesterase and butyrylcholinesterase. The basic extract was after an initial extraction with 95% ethanol pre-purified by extraction with chloroform. This chloroform extract was divided further by column chromatography on a total of 531 fractions. The fractions obtained were, based on the similarities identified by thin layer chromatography, interconnected in a final number of 17 fractions. The mobile phase for column chromatography were mixtures of chloroform and medical gasoline, chloroform and ethanol, and ethanol itself. For further testing were used fractions number 2 and 5. From the fraction 2 was by preparative thin layer chromatography and subsequent crystallization isolated substance number 1. After analysis of this substance by GC/MS was found that it is a (+) - vincaminorein. Further was examined it's biological activity towards cholinesterases, which found a...

The genus Aspidosperma Mart. & Zucc. is often used for its wood in the Amazon. This genus is taxonomically complex because of morphologically similar species. Anatomy is a resource for distinguishing these taxa. The objective of this study was to anatomically characterize the leaves and wood of Aspidosperma excelsum, A. carapanauba, A. desmanthum, and A. spruceanum, as a contribuition to their identification. Standard wood and leaf anatomical techniques were used. The studied species showed similarities for: epicuticular waxes, thick cuticle, bicolatoral vascular bundles, filiform trichomes, straight-sided epidermal cells pentagonal to hexagonal. Differences were seen in cuticular ornamentation, organization of vascular bundles in pedicles, and in leaf architecture, such that two groups can be formed. Wood of the studied species is quite similar, having the following characteristics in common: homogeneous rays uniseriate to triseriate, small and diffuse solitary pores. The principal structure for distinguishing these species is the axial parenchyma that varies from diffuse apotracheal to aliform paratracheal with losangular extention with short aleta. These anatomical structures, both of the leaves and the wood, are sufficient to distinguish the studied species. / O gênero Aspidosperma Mart.& Zucc. é freqüentemente utilizado na região Amazônica, principalmente no mercado madeireiro, entretanto, o referido gênero apresenta uma grande complexidade taxonômica, devido às semelhanças morfológicas de suas espécies. A anatomia pode ser um recurso para auxiliar a distinção destes taxa. Com isso, objetivou-se no presente trabalho caracterizar anatomicamente a folha e a madeira de Aspidosperma excelsum, A. carapanauba, A. desmanthum e A. spruceanum, contribuindo com a identificação das mesmas. Para alcançar tal objetivo, realizaram-se técnicas usuais em anatomia tanto para folha quanto para a madeira. As espécies estudadas apresentam semelhanças para: ceras epicuticulares, cutícula espessada, feixes vasculares bicolaterais, tricomas filiformes, células epidérmicas com paredes retas, pentagonais a hexagonais. Já as diferenças foram quanto à ornamentação da cutícula, organização dos feixes vasculares no pecíolo e na arquitetura foliar que proporcionam a formação de dois grupos. A madeira das referidas espécies são bastante semelhante macroscopicamente, tendo como características comuns: os raios homogêneos unisseriados a trisseriados, poros solitários, pequenos e difusos, contudo, a principal estrutura para distinção das espécies é o parênquima axial, variando de apotraqueal difuso a paratraqueal aliforme de extensão losangular com aleta curta. De posse desses resultados, verifica-se que as estruturas anatômicas, tanto da folha quanto da madeira, podem distinguir as espécies estudadas.

Šípková, P.: Biological activity of secondary plants metabolites X. Alkaloids of Vinca minor L. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2019, 66 s. Key words: Apocynaceae, Vinca minor, alkaloids, isolation, biological activity, screening Alzheimer's disease is a progressive neurodegenerative disease. The number of affected patients is constantly increasing. This disease cannot be treated casually, therefore discovering and testing new substances that could potentially be used in a treatment is very important. The Vinca minor L. fraction after column chromatography was separated by flash chromatography. Isolation of the individual alkaloids was performed by preparative TLC. Based on NMR and MS analyses and comparison with literature, alkaloids were identified as vincarubine and (-)-vinoxine. Modified Ellman's method was used to test cholinesterase inhibitory activity of isolated alkaloids. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are enzymes, that play a very important role in the pathofysiology of Alzheimer's diasease. (-)-Vinoxine showed relatively high activity against BuChE (IC50 = 24,61 ± 1,71 µM), inhibitory activity against AChE was insignificant (IC50 > 1000µM). Vincarubin did not...

Marečková, L .: Biological activity of secondary plant metabolites VII. Alkaloids of Vinca minor L. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Kralove, Hradec Králové, Department of pharmaceutical botany and ecology, Hradec Králové 2018 There was prepared an extract from aerial parts of the plant Vinca minor L. This prepared extract was separated by the column chromatography into individual fractions. One chosen fraction was separated by TLC on silicagel. Two substances were isolated and they we identified by GC/MS and NMR as (-)-minovin and (+)-minovincin. These two substances were tested for their inhibition activity against human acetylcholinesterase (hemolysate of human erythrocytes) and butyrylcholinesterase (human plasma). Their inhibition activity was compared to standards which were Galanthamin, Huperzin A and Eserin. (+)-Minovincin did not show significant inhibitory activity against either enzyme (IC50 = AchE > 1000 µM, BuChE 655,05 ± 35,37 µM) and it is not suitable for further investigation. (-)-Minovin showed no significant activity against AChE (IC50 = 234,27 ± 63,40 µM), activity against BuChE (IC50 = 26,32 ± 2,52 µM) was higher than inhibitory akctivity of Galanthamin, but still relatively low for further investigation. The aim of this work was mainly the...

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