Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O cÃncer à uma complexa doenÃa de origem genÃtica, resultante de mutaÃÃes em proto-oncogenes e/ou genes supressores tumorais. à considerada uma das principais causas de morte por doenÃa no mundo. Estudos preliminares com o extrato acetÃnico e compostos isolados da espÃcie Annona muricata evidenciaram o seu potencial antitumoral. Annona muricata L. (Annonaceae) à popularmente conhecida como gravioleira; possui ampla distribuiÃÃo, principalmente em regiÃes tropicais. No Brasil, a espÃcie à utilizada como analgÃsico, hipoglicemiante, anti-inflamatÃrio, antitÃrmico, hepatoprotetor, antinevrÃlgico, antiparasitÃrio e antirreumÃtico. O presente trabalho descreve o isolamento de uma acetogenina, denominada anonacina, com anel mono-tetrahidrofurano, fÃrmula molecular C35H64O7 e massa de 596 u.m.a à partir do extrato acetÃnico das folhas da referida espÃcie, atravÃs de prospecÃÃo quÃmica bioguiada pela citotoxicidade usando o ensaio do MTT. Todas as fraÃÃes obtidas e a acetogenina isolada na prospecÃÃo apresentaram potencial citotÃxico nas linhagens tumorais testadas. Os valores de concentraÃÃo inibitÃria mÃdia dessa acetogenina, apresentaram-se na ordem de nano-molar. O ensaio de citotoxicidade mostrou que esse composto apresenta atividade antiproliferativa frente à todas as linhagens tumorais testadas, com CI50 variando de 3.280 nM a 1,56 nM em HL-60 e HCT-116, respectivamente apÃs 72 horas de incubaÃÃo, onde a substÃncia foi mais ativa frente Ãs linhagens HCT-116, OVCAR-8, SF-295 e PC-3M. Essa acetogenina nÃo foi capaz de inibir a proliferaÃÃo de cÃlulas mononucleares do sangue perifÃrico, embora tenha apresentado efeito hemolÃtico em eritrÃcitos de camundongos. A acetogenina AMFA-4, em linhagem de cÃlula de cÃncer colorretal, apresentou efeito citotÃxico dependente da dose e do tempo de incubaÃÃo, sendo capaz de alterar o nÃmero de cÃlulas viÃveis e nÃo-viÃveis apÃs 48 e 72 h de exposiÃÃo. Nesses dois tempos de incubaÃÃo, essa molÃcula foi capaz de alterar a morfologia das cÃlulas HCT-116, onde a mesma teve aumento do nÃcleo e citoplasma e aspecto fusiforme. O presente trabalho mostrou que o extrato acetÃnico das folhas de A. muricata à uma fonte promissora para obtenÃÃo de compostos pertencentes a classe das acetogeninas com atividade citotÃxica relevante em cÃlulas tumorais de origem humana. / Cancer is a complex disease of genetic origin, resulting from mutations in proto-oncogenes and / or tumor suppressor genes. It is considered one of the major causes of death in the world. Preliminary studies with the acetone extract and isolated compounds from the Annona muricata species demonstrated their antitumor potential. Annona muricata L. (Annonaceae), popularly known as soursop, has wide distribution, mainly in the tropical regions. In Brazil, the species is widely used as analgesic, hypoglycemic, anti-inflammatory, antipyretic, hepatoprotective, anti-neuralgic, anti-parasitic and anti-rheumatic. This study reports the isolation of an acetogenin, called Annonacin, with a mono-tetrahydrofuran ring, the molecular formula: C35H64O7 and a mass of 596 u.m.a from the acetone extract of Annona muricata leaves through a bioguided chemical prospecting by cytotoxicity using the MTT assay. All the obtained fractions and the isolated acetogenin showed a cytotoxic potential in the tested tumor cell lines. The values of the mean inhibitory concentration of this acetogenin, had an order of nano-molar. The cytotoxicity assay demonstrated that the compound has anti-proliferative activity against all the tested tumor cell lines, with an IC50 ranging from 3.280 nM to 1.56 nM in HL-60 and HCT-116, respectively, after 72 hours of incubation, where the substance was most active against the following tumor cell lines: HCT-116, OVCAR-8, SF-295 and PC-3M. This acetogenin was not able to inhibit the proliferation of normal peripheral blood cells, although it showed a hemolytic effect on mice erythrocytes. The Annonacin, in a colorectal cancer cell line, showed a cytotoxic effect strongly related to the dose and incubation time, being able to change the number of viable and non-viable cells after 48 and 72 h of exposure. In both incubation times, this molecule was able to modify the HCT-116 cell morphology, where it had an increase on the cytoplasm and nucleus size, besides a fusiform appearance. This study showed that the acetone extract from the A. muricata leaves is a promising source for obtaining compounds belonging to the acetogenins class with significant cytotoxic activity on tumor cells of human origin.

多年來，儘可能多的從植物中提取單體化合物一直是開發新型化學防癌劑和化學治療劑藥物的重要來源。 / 在本课题中，我们活性測試了从刺果紫玉盘（番荔枝科植物）中分离得到的14个番荔枝内酯化合物和7个多氧环己烯化合物，从三叉刺（豆科植物）和黄瑞香（瑞香科植物）中分离得到的4个黄酮化合物，从黄瑞香（瑞香科植物）和了哥王（瑞香科植物）中分离得到的2个香豆素化合物，以及从总状蕨藻（蕨藻科植物）中分离得到的1 个生物碱化合物，對11種人類常見癌症細胞株，如惡性黑色素瘤、肺癌、子宮頸上皮腺癌、肝癌、前列腺癌、結直腸癌的體外抗癌活性，用以建立一個全面的抗癌活性數據庫，為人們更好得了解番荔枝科植物奠定基礎。 / 在這些被篩選的單體化合物中，番荔枝內酯（ACGs）顯示出卓越的抗癌活性。它們對某些癌細胞株的細胞毒性甚至達到了nmol/l級別。例如番荔枝內酯desacetyluvaricin（Dau），對11條人類癌細胞株具有廣泛的抗增生活性，其半抑制濃度（IC₅₀）範圍從2.3 nM到37.4 μM。其中，Dau對結直腸癌細胞SW480的毒性最甚。Dau不僅具有高的抗癌效力，并對人正常纖維細胞Hs68幾乎沒有細胞毒性，半抑制濃度超過了247.5 μM。進一步的機理研究表明，Dau可導致SW480細胞產生大量過氧化物，進而導致細胞核內DNA斷裂。DNA損傷會讓MEK/ERK信號通路失活，並且影響了細胞週期調控蛋白的正常表達。例如影響細胞S週期的調控蛋白Cyclin A和Cyclin E的表達，以及影響G₁/S檢查點調控蛋白E2F的表達。由此，Dau促進SW480癌細胞穿過G₁/S檢查點，由G₁進入S期並在S期累計。最終被抑制在S週期的SW480細胞發生了壞死。以上機理的研究可為更好的理解ACG的作用機制提供一定的理論基礎。 / 番荔枝內酯是一系列長鏈脂肪酸的衍生物。它的結構的多樣性引發了我們極大的興趣去研究它的構效關係。我們比較了14個番荔枝內酯在細胞毒性和細胞週期控制方面對兩種不同的前列腺癌細胞LNCaP（p53基因野生型）和PC-3（p53基因缺失型）的影響。實驗結果表明，LNCaP細胞比PC-3更加敏感。番荔枝內酯的這種選擇性大概跟癌細胞中p53抑癌蛋白的表達水平有關。此外，關於構效關係的研究我們還發現：（1）在番荔枝內酯結構的核心系統中，四氫呋喃環的個數越多，化合物的抗癌活性越高；（2）在含有相鄰雙四氫呋喃環結構的化合物中，擁有threo/trans/threo/trans/erythro立體構型的化合物的細胞毒性比擁有threo/trans/threo/trans/threo立體構型的化合物高；（3）含單或雙四氫呋喃環結構的番荔枝內酯都將通過將LNCaP細胞抑制在G₁/G₀週期從而達到抗癌效果，並不會引起細胞凋亡；（4）含單四氫呋喃環結構的番荔枝內酯都將通過引發細胞凋亡從而達到抑制PC-3癌細胞的增長。然而含雙四氫呋喃環結構的番荔枝內酯會引發更多的PC-3細胞凋亡，並且有不同程度的細胞週期抑制；（5）在四氫呋喃環核心體系上，乙酰氧基會比羥基增加番荔枝內酯的細胞毒性；（6）雙鍵的取代基也會增加毒性效果。我們的研究結果印證了一些文獻已報導的關於番荔枝內酯構效關係的結論，同時我們也提出了一些新的假設。 / 本研究不僅增加了我們對番荔枝內酯強大的抗癌活性更全面的了解，並且通過機理研究還為它的選擇性毒性及構效關係特點提供了有重要的信息。番荔枝內酯是一類具有充滿前景抗癌化合物。在接下來的研究中，我們將致力於體內抗癌活性的研究，并擴大研究範圍，通過對多個ACG化合物的機理研究來證明我們對它的選擇性毒性的機理假設。 / For years and years, the discovery of phytochemicals as many as possible has always been an important strategy for the development of novel chemopreventive and chemotherapeutic drugs. / In this studies, we have screened 14 Annonaceous acetogenins and 7 polyoxygenated cyclohexenes isolated from the root of Uvaria calamistrata (Annonaceae), 4 flavonoids isolated from the stems of Trifidacanthus unifoliolatus (Fabaceae) and Daphne giraldii (Thymelaeaceae), 2 cumarins isolated from the stem bark of Daphne giraldii (Thymelaeaceae) and the root of Wikstroemia indica (Thymelaeaceae), and 1 alkaloid isolated from Caulerpa racemosa (Caulerpaceae). The in vitro anticancer effects of these 28 natural compounds on 11 human cancer cell lines, including malignant melanoma, lung carcinoma, cervix epithelial adenocarcinoma, liver carcinoma, prostate adenocarcinoma and colorectal adenocarcinoma, were tested to set up an overall anticancer activity database for better understanding of the biological actions of Annonaceous plants. / Among the screened natural compounds, Annonaceous acetogenins (ACGs) exhibited outstanding anticancer efficacy. The cytotoxicities of ACGs to some cancer cell lines were even at nmol/l level. For instance, desacetyluvaricin (Dau), an ACG, was identified as a novel antiproliferative agent with a broad spectrum of inhibitions against the tested 11 human cancer cell lines with the IC₅₀ values ranging from 2.3 nM to 37.4 μM, and was especially cytotoxic to SW480 human colorectal carcinoma cells. Despite this potency, Dau was virtually nontoxic toward Hs68 human fibroblasts with an IC₅₀ value exceeding 247.5 μM. Further cell death mechanism studies showed that Dau could induce large amounts of superoxide production, which subsequently induced nuclear DNA fragmentation. DNA damage may inactivate the MEK/ERK signaling pathway and disturbed the expressions of cell cycle regulators such as Cyclin A and Cyclin E, which are S phase regulators, and E2F which is the G1/S checkpoint regulator. Thereafter, Dau arrested SW480 cells in S phase by promoting SW480 cells passing through the G₁/S boundary, and then accumulating in S phase. Finally, the SW480 cells underwent necrotic cell death. This mechanism study may provide a better understanding on the action mode of ACGs. / ACGs are derivatives of long chain fatty acids. Its structural diversity kindled our great interests in its structure-activity relationship (SAR). Therefore, we compared the cytotoxicities and cell cycle regulations of the 14 ACG compounds on two different human prostate cancer cell lines, LNCaP (p53 wild-type) and PC-3 (p53 null-type). Results showed that LNCaP cells were more sensitive to ACGs than PC-3 cells. This selectivity may be due to the presence of p53 tumor suppressor gene. Moreover, we found about SAR study that (1) the more THF rings existing in the core structure of ACGs, the more potent anticancer effects of ACGs would be; (2) for the adjacent bis-THF ACGs, stereo-structure with threo/trans/threo/trans/erythro configuration is generally more cytotoxic than the one with threo/trans/threo/trans/threo configuration; (3) both mono-THF ACGs and bis-THF ACGs inhibited LNCaP cells growth by G₁/G₀ phase arrest without any apoptosis induction; (4) mono-THF ACGs inhibited PC-3 cells growth by inducing apoptosis without cell cycle disturbance. However, the bis-THF ACGs could induce more apoptosis in PC-3 cells with partially cell cycle arrest. (5) the -OAc substituent group instead of -OH in the THF system would enhance the cytotoxicity efficacies of ACGs; (6) the double bond substituent would also enhance the anticancer effect. Our studies have proved several reported disciplines about the SAR of ACGs, and also proposed some new hypothesis. / Taken together, this study not only increased our understanding on the potent anticancer effects of ACG, but also provided valuable information on explaining its special cytotoxicities and the SAR properties through underling mechanism study. ACGs are a group of promising anticancer compounds with potent and steady activities. In the future work, we should further examine the in vivo anticancer effects and study more ACGs on their action modes to validate our hypothesis on their sensitivities to certain cancer cell lines. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xue, Junyi. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 215-236). / Abstracts also in Chinese.

To investigate potential sources of novel grain protectors, this study evaluated, firstly, the bioactivity of ethanolic extracts (66) prepared from 29 species belonging to 11 different genera of Neotropical Annonaceae against the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae). A screening assay demonstrated that the most pronounced effects (acute and chronic) on S. zeamais were caused by extracts from the Annona montana, A. mucosa, A. muricata and A. sylvatica seeds, and, to a lesser extent, by extracts prepared from leaves of A. montana, A. mucosa, A. muricata, and Duguetia lanceolata. However, the most active extracts (from seeds) did not affect fungal growth and aflatoxin production of Aspergillus flavus (Ascomycota: Trichocomaceae). Using the maize weevil as bioindicator, bioguided fractionations were then conducted in order to isolate, purify and characterize the possible active compound(s) from the most interesting extracts. By means of different chromatographic procedures, nine compounds (five acetogenins, three steroids, and one aromatic compound) were isolated. The acetogenins rolliniastatin-1 and ACG4 (structural determination in progress) and the aromatic compound 2,4,5- trimethoxystyrene as well as the steroids campesterol, stigmasterol, and sitosterol [tested in mixture (8.44 + 12.37 + 79.19%, respectively)] showed promising grain protective properties. Furthermore, the obtained results indicate that compounds from different chemical natures have a synergistic effect on the overall biological activity of the crude extracts. In a second study, the acute and chronic toxicity of selected ethanolic seed extracts from Annona species (A. montana, A. mucosa, A. muricata, and A. sylvatica) and an acetogenin-based commercial bioinsecticide (Anosom® 1EC) were investigated against the cabbage looper Trichoplusia ni (Lepidoptera: Noctuidae) and the green peach aphid Myzus persicae (Hemiptera: Aphididae). In the laboratory, extracts of A. mucosa and A. sylvatica as well as Anosom® were especially active through oral and topical administration. A greenhouse trial showed that a formulated A. mucosa extract and Anosom® were highly effective (>98% mortality) against third instar of T. ni larvae, and comparable to a pyrethrin-based commercial insecticide (Insect Spray®) used as a positive control. Similar to results with T. ni, A. mucosa extract showed the greatest aphicidal either in laboratory or greenhouse bioassays. In a third study, the acaricidal activity [against the citrus red mite Panonychus citri (Acari: Tetranychidae)] of the ethanolic extract from A. mucosa seeds (most active) was investigated. In laboratory tests, it exhibited levels of activity superior to commercial acaricides/insecticides of natural origin [Anosom® 1EC (annonin), Derisom® 2EC (karanjin), and Azamax® 1.2EC (azadirachtin + 3- tigloylazadirachtol)] and similar to a synthetic acaricide [Envidor® 24 SC (spirodiclofen)]. Finally, the compatibility of A. mucosa seed extract with three entomopathogenic fungi species (Beauveria bassiana, Isaria fumosorosea and Metarhizium anisopliae) was assessed. In overall, it was compatible with the three entomopathogenic fungi species when tested at recommended concentrations for target pest species control. Therefore, this study argues for the use of derivatives from Neotropical Annonaceae as a useful component in the framework of integrated pest management (IPM) programs. / Visando investigar potenciais fontes de novos protetores de grãos, este estudo avaliou, primeiramente, a bioatividade de extratos etanólicos (66) obtidos de 29 espécies pertencentes a 11 diferentes gêneros de anonáceas neotropicais sobre o gorgulho-do-milho Sitophilus zeamais (Coleoptera: Curculionidae). A triagem inicial demonstrou que os efeitos (agudos e crônicos) mais pronunciados sobre S. zeamais foram causados pelos extratos de sementes de Annona montana, de A. mucosa, de A. muricata e de A. sylvatica, seguidos pelos extratos de folhas de A. montana, de A. mucosa, de A. muricata e de Duguetia lanceolata. No entanto, os extratos mais ativos (sementes) não afetaram o crescimento vegetativo e a produção de aflatoxinas de um isolado de Aspergillus flavus (Ascomycota: Trichocomaceae). Fracionamentos biomonitorados foram então realizados a fim de isolar, purificar e caracterizar o(s) composto(s) ativo(s) majoritário(s) dos extratos mais promissores, utilizando-se, para isso, o gorgulho-do-milho como bioindicador. Por meio de diferentes procedimentos cromatográficos, foram isolados nove compostos: cinco acetogeninas, três esteroides e um composto aromático. As acetogeninas roliniastatina-1 e ACG4 (determinação estrutural em andamento), o composto aromático 2,4,5-trimetoxiestireno e os esteroides campesterol, estigmasterol e sitosterol [testados em mistura (8,44 + 12,37 + 79,19%, respectivamente)] mostraram promissoras propriedades protetoras de grãos. Em geral, os resultados obtidos indicaram que compostos de diferentes naturezas químicas têm efeito sinérgico sobre a atividade biológica dos extratos brutos. No segundo estudo, foi avaliada a toxicidade aguda e crônica dos extratos selecionados de sementes de Annona (A. montana, A. mucosa, A. muricata e A. sylvatica) e de um bioinseticida comercial à base de acetogeninas (Anosom® 1EC) sobre a lagarta-mede-palmo Trichoplusia ni (Lepidoptera: Noctuidae) e sobre o pulgão-verde Myzus persicae (Hemiptera: Aphididae). Em laboratório, os extratos de A. mucosa e de A. sylvatica e o bioinseticida Anosom® foram especialmente ativos através da administração oral e tópica. Em casa de vegetação, um extrato formulado de A. mucosa e Anosom® foram altamente eficazes contra larvas de terceiro ínstar de T. ni, com eficácia comparável ao de um inseticida comercial à base de piretrinas (Insect Spray®) utilizado como controle positivo. Similar aos resultados com T. ni, o extrato de A. mucosa apresentou a maior atividade aficida, tanto em bioensaios em laboratório quanto em casa de vegetação. No terceiro estudo, a atividade acaricida [sobre o ácaro-purpúreo-dos-citros Panonychus citri (Acari: Tetranychidae)] do extrato de sementes de A. mucosa (mais ativo) foi avaliada em bioensaios laboratoriais. O extrato de A. mucosa apresentou eficácia superior aos acaricidas/inseticidas comerciais de origem natural [Anosom® 1EC (anonina), Derisom® 2EC (karanjina) e Azamax® 1.2EC (azadiractina + 3-tigloilazadiractol)] e similar a um acaricida sintético [Envidor® 24SC (espirodiclofeno)]. Finalmente, foi avaliada a compatibilidade do extrato de sementes de A. mucosa com três espécies de fungos entomopatogênicos (Beauveria bassiana, Isaria fumosorosea e Metarhizium anisopliae). De modo geral, o extrato de A. mucosa foi compatível com as três espécies quando testado nas concentrações preconizadas para o controle das espécies-praga alvo. Assim, este estudo fornece importantes subsídios para o uso de derivados de anonáceas neotropicais como um componente útil para os programas de manejo integrado de pragas (MIP).

Visando detectar alternativas de manejo para a lagarta-do-cartucho-do-milho, Spodoptera frugiperda (Lepidoptera: Noctuidae), realizou-se, primeiramente, uma triagem em extratos etanólicos preparados das estruturas (folhas, ramos e sementes) de diferentes espécies de Annonaceae (Annona cacans, A. montana, A. mucosa, A. reticulata, A. sylvatica e Duguetia lanceolata). Com base nessa etapa inicial, constatou-se que o extrato etanólico das sementes de A. mucosa (ESAM) foi o mais promissor, causando significativa toxicidade aguda (CL50 e CL90 de 842,97 e 1.882,00 mg kg-1, respectivamente) e pronunciada inibição do desenvolvimento larval (toxicidade crônica), após sete dias de exposição a dietas tratadas. Na CL50, o ESAM reduziu as viabilidades larval e pupal e o peso de pupas com 24 horas, e aumentou a duração da fase larval. No entanto, o ESAM não ocasionou efeito fagodeterrente para larvas de quarto ínstar de S. frugiperda, embora tenha reduzido o consumo ao longo do tempo. Feito isso, a eficácia de ESAM foi comparada com inseticidas comerciais de origem natural (Azamax® 1,2 EC) e sintética (Premio® SC). Nessa comparação, o ESAM (na CL90) apresentou eficácia similar (equitóxico) aos dois produtos comerciais (utilizados na dose registrada para o controle do inseto-praga). A seguir, foi realizado o fracionamento biomonitorado através de diferentes técnicas cromatográficas que conduziu ao isolamento da acetogenina roliniastatina-1, identificada (com base em técnicas espectroscópicas) como componente majoritário da fração mais ativa do extrato etanólico de A. mucosa. Roliniastatina-1 foi então novamente ensaiada frente a larvas de S. frugiperda e ocasionou significativos efeitos agudos (mortalidade larval) e crônicos (redução do desevolvimento larval). Por fim, investigou-se a bioatividade de uma formulação à base de acetogeninas (Anosom® 1 EC) recentemente registrada, tanto isoladamente quanto em mistura com uma formulação à base de limonoides (Azamax® 1,2 EC). Anosom® 1 EC, testado na CL90 estimada (2.959,00 mg kg-1) e Azamax® 1,2 EC, testado na concentração registrada para o controle do inseto-praga (4.000,00 mg kg-1), causaram significativa mortalidade larval, sem ocorrer diferença entre os tratamentos tanto quando testados isoladamente como em mistura binária. Anosom® 1 EC (na CL50) provocou ainda aumento significativo das mortalidades larval e pupal e da duração da fase larval, bem como redução do peso pupal do inseto. Dessa forma, derivados de Annonaceae podem constituir um componente útil para o manejo integrado de S. frugiperda em condições de campo. / A screening with ethanolic extracts prepared from structures (leaves, branches and seeds) from different species of Annonaceae (Annona cacans, A. montana, A. mucosa, A. reticulate, A. sylvatica and Duguetia lanceolata) aiming to detect management alternatives for Spodoptera frugiperda (Lepidoptera: Noctuidae) was done. Based on this initial phase, it was verified that the ethanolic extract from seeds of A. mucosa (AMSE) was the most promising one, causing significant acute toxicity (LC50 and LC90 of 842.97 and 1,822.00 mg kg-1, respectively) and pronounced inhibition of larval development (chronic toxicity) after a 7-day exposition to treated diets. At LC50, AMSE decreased larval and pupal viabilities and also the pupal weight at 24 hours, and increased the larval phase duration. However, AMSE didn\'t cause antifeedant effect for 4-instar S. frugiperda larvae, though it decreased consumption throughout the time. After that, AMSE efficacy was compared to commercial pesticides from natural (Azamax® 1.2 EC) and synthetic (Premio® SC) origin. In this confrontation, AMSE (at LC90) showed efficacy similar to that of commercial products (used at registered dose for S. frugiperda control). Then, the bioguided fractionation using different chromatographic techniques which led to isolation of acetogenin rolliniastatin-1 identified as the majoritary compound of the most active ethanolic extract from A. mucosa. After that, rolliniastatin-1 was again tested against S. frugiperda larvae and caused significant acute (larval mortality) and chronic (decrease of larval development) effects. Finally, the bioactivity of a newly-registered acetogenin-based (Anosom® 1 EC) formulation was investigated, both separately and mixed with a limonoid-based formulation (Azamax® 1.2 EC). Anosom® 1 EC, tested at estimated LC90 (2,959.00 mg kg-1) and Azamax® 1.2 EC, tested at registered dose for S. frugiperda (4,000.00 mg kg-1) led to significant larval mortality, without happening any differences between treatments either tested separately or in a binary mixture. Anosom® 1 EC (at LC50) also led to significant increase of larval and pupal mortality and larval phase, as well as insect pupal weight decrease. Thus, Annonaceae derivates can be a useful component for S. frugiperda integrated management under field conditions.

To investigate potential sources of novel grain protectors, this study evaluated, firstly, the bioactivity of ethanolic extracts (66) prepared from 29 species belonging to 11 different genera of Neotropical Annonaceae against the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae). A screening assay demonstrated that the most pronounced effects (acute and chronic) on S. zeamais were caused by extracts from the Annona montana, A. mucosa, A. muricata and A. sylvatica seeds, and, to a lesser extent, by extracts prepared from leaves of A. montana, A. mucosa, A. muricata, and Duguetia lanceolata. However, the most active extracts (from seeds) did not affect fungal growth and aflatoxin production of Aspergillus flavus (Ascomycota: Trichocomaceae). Using the maize weevil as bioindicator, bioguided fractionations were then conducted in order to isolate, purify and characterize the possible active compound(s) from the most interesting extracts. By means of different chromatographic procedures, nine compounds (five acetogenins, three steroids, and one aromatic compound) were isolated. The acetogenins rolliniastatin-1 and ACG4 (structural determination in progress) and the aromatic compound 2,4,5- trimethoxystyrene as well as the steroids campesterol, stigmasterol, and sitosterol [tested in mixture (8.44 + 12.37 + 79.19%, respectively)] showed promising grain protective properties. Furthermore, the obtained results indicate that compounds from different chemical natures have a synergistic effect on the overall biological activity of the crude extracts. In a second study, the acute and chronic toxicity of selected ethanolic seed extracts from Annona species (A. montana, A. mucosa, A. muricata, and A. sylvatica) and an acetogenin-based commercial bioinsecticide (Anosom® 1EC) were investigated against the cabbage looper Trichoplusia ni (Lepidoptera: Noctuidae) and the green peach aphid Myzus persicae (Hemiptera: Aphididae). In the laboratory, extracts of A. mucosa and A. sylvatica as well as Anosom® were especially active through oral and topical administration. A greenhouse trial showed that a formulated A. mucosa extract and Anosom® were highly effective (>98% mortality) against third instar of T. ni larvae, and comparable to a pyrethrin-based commercial insecticide (Insect Spray®) used as a positive control. Similar to results with T. ni, A. mucosa extract showed the greatest aphicidal either in laboratory or greenhouse bioassays. In a third study, the acaricidal activity [against the citrus red mite Panonychus citri (Acari: Tetranychidae)] of the ethanolic extract from A. mucosa seeds (most active) was investigated. In laboratory tests, it exhibited levels of activity superior to commercial acaricides/insecticides of natural origin [Anosom® 1EC (annonin), Derisom® 2EC (karanjin), and Azamax® 1.2EC (azadirachtin + 3- tigloylazadirachtol)] and similar to a synthetic acaricide [Envidor® 24 SC (spirodiclofen)]. Finally, the compatibility of A. mucosa seed extract with three entomopathogenic fungi species (Beauveria bassiana, Isaria fumosorosea and Metarhizium anisopliae) was assessed. In overall, it was compatible with the three entomopathogenic fungi species when tested at recommended concentrations for target pest species control. Therefore, this study argues for the use of derivatives from Neotropical Annonaceae as a useful component in the framework of integrated pest management (IPM) programs. / Visando investigar potenciais fontes de novos protetores de grãos, este estudo avaliou, primeiramente, a bioatividade de extratos etanólicos (66) obtidos de 29 espécies pertencentes a 11 diferentes gêneros de anonáceas neotropicais sobre o gorgulho-do-milho Sitophilus zeamais (Coleoptera: Curculionidae). A triagem inicial demonstrou que os efeitos (agudos e crônicos) mais pronunciados sobre S. zeamais foram causados pelos extratos de sementes de Annona montana, de A. mucosa, de A. muricata e de A. sylvatica, seguidos pelos extratos de folhas de A. montana, de A. mucosa, de A. muricata e de Duguetia lanceolata. No entanto, os extratos mais ativos (sementes) não afetaram o crescimento vegetativo e a produção de aflatoxinas de um isolado de Aspergillus flavus (Ascomycota: Trichocomaceae). Fracionamentos biomonitorados foram então realizados a fim de isolar, purificar e caracterizar o(s) composto(s) ativo(s) majoritário(s) dos extratos mais promissores, utilizando-se, para isso, o gorgulho-do-milho como bioindicador. Por meio de diferentes procedimentos cromatográficos, foram isolados nove compostos: cinco acetogeninas, três esteroides e um composto aromático. As acetogeninas roliniastatina-1 e ACG4 (determinação estrutural em andamento), o composto aromático 2,4,5-trimetoxiestireno e os esteroides campesterol, estigmasterol e sitosterol [testados em mistura (8,44 + 12,37 + 79,19%, respectivamente)] mostraram promissoras propriedades protetoras de grãos. Em geral, os resultados obtidos indicaram que compostos de diferentes naturezas químicas têm efeito sinérgico sobre a atividade biológica dos extratos brutos. No segundo estudo, foi avaliada a toxicidade aguda e crônica dos extratos selecionados de sementes de Annona (A. montana, A. mucosa, A. muricata e A. sylvatica) e de um bioinseticida comercial à base de acetogeninas (Anosom® 1EC) sobre a lagarta-mede-palmo Trichoplusia ni (Lepidoptera: Noctuidae) e sobre o pulgão-verde Myzus persicae (Hemiptera: Aphididae). Em laboratório, os extratos de A. mucosa e de A. sylvatica e o bioinseticida Anosom® foram especialmente ativos através da administração oral e tópica. Em casa de vegetação, um extrato formulado de A. mucosa e Anosom® foram altamente eficazes contra larvas de terceiro ínstar de T. ni, com eficácia comparável ao de um inseticida comercial à base de piretrinas (Insect Spray®) utilizado como controle positivo. Similar aos resultados com T. ni, o extrato de A. mucosa apresentou a maior atividade aficida, tanto em bioensaios em laboratório quanto em casa de vegetação. No terceiro estudo, a atividade acaricida [sobre o ácaro-purpúreo-dos-citros Panonychus citri (Acari: Tetranychidae)] do extrato de sementes de A. mucosa (mais ativo) foi avaliada em bioensaios laboratoriais. O extrato de A. mucosa apresentou eficácia superior aos acaricidas/inseticidas comerciais de origem natural [Anosom® 1EC (anonina), Derisom® 2EC (karanjina) e Azamax® 1.2EC (azadiractina + 3-tigloilazadiractol)] e similar a um acaricida sintético [Envidor® 24SC (espirodiclofeno)]. Finalmente, foi avaliada a compatibilidade do extrato de sementes de A. mucosa com três espécies de fungos entomopatogênicos (Beauveria bassiana, Isaria fumosorosea e Metarhizium anisopliae). De modo geral, o extrato de A. mucosa foi compatível com as três espécies quando testado nas concentrações preconizadas para o controle das espécies-praga alvo. Assim, este estudo fornece importantes subsídios para o uso de derivados de anonáceas neotropicais como um componente útil para os programas de manejo integrado de pragas (MIP).

Visando detectar alternativas de manejo para a lagarta-do-cartucho-do-milho, Spodoptera frugiperda (Lepidoptera: Noctuidae), realizou-se, primeiramente, uma triagem em extratos etanólicos preparados das estruturas (folhas, ramos e sementes) de diferentes espécies de Annonaceae (Annona cacans, A. montana, A. mucosa, A. reticulata, A. sylvatica e Duguetia lanceolata). Com base nessa etapa inicial, constatou-se que o extrato etanólico das sementes de A. mucosa (ESAM) foi o mais promissor, causando significativa toxicidade aguda (CL50 e CL90 de 842,97 e 1.882,00 mg kg-1, respectivamente) e pronunciada inibição do desenvolvimento larval (toxicidade crônica), após sete dias de exposição a dietas tratadas. Na CL50, o ESAM reduziu as viabilidades larval e pupal e o peso de pupas com 24 horas, e aumentou a duração da fase larval. No entanto, o ESAM não ocasionou efeito fagodeterrente para larvas de quarto ínstar de S. frugiperda, embora tenha reduzido o consumo ao longo do tempo. Feito isso, a eficácia de ESAM foi comparada com inseticidas comerciais de origem natural (Azamax® 1,2 EC) e sintética (Premio® SC). Nessa comparação, o ESAM (na CL90) apresentou eficácia similar (equitóxico) aos dois produtos comerciais (utilizados na dose registrada para o controle do inseto-praga). A seguir, foi realizado o fracionamento biomonitorado através de diferentes técnicas cromatográficas que conduziu ao isolamento da acetogenina roliniastatina-1, identificada (com base em técnicas espectroscópicas) como componente majoritário da fração mais ativa do extrato etanólico de A. mucosa. Roliniastatina-1 foi então novamente ensaiada frente a larvas de S. frugiperda e ocasionou significativos efeitos agudos (mortalidade larval) e crônicos (redução do desevolvimento larval). Por fim, investigou-se a bioatividade de uma formulação à base de acetogeninas (Anosom® 1 EC) recentemente registrada, tanto isoladamente quanto em mistura com uma formulação à base de limonoides (Azamax® 1,2 EC). Anosom® 1 EC, testado na CL90 estimada (2.959,00 mg kg-1) e Azamax® 1,2 EC, testado na concentração registrada para o controle do inseto-praga (4.000,00 mg kg-1), causaram significativa mortalidade larval, sem ocorrer diferença entre os tratamentos tanto quando testados isoladamente como em mistura binária. Anosom® 1 EC (na CL50) provocou ainda aumento significativo das mortalidades larval e pupal e da duração da fase larval, bem como redução do peso pupal do inseto. Dessa forma, derivados de Annonaceae podem constituir um componente útil para o manejo integrado de S. frugiperda em condições de campo. / A screening with ethanolic extracts prepared from structures (leaves, branches and seeds) from different species of Annonaceae (Annona cacans, A. montana, A. mucosa, A. reticulate, A. sylvatica and Duguetia lanceolata) aiming to detect management alternatives for Spodoptera frugiperda (Lepidoptera: Noctuidae) was done. Based on this initial phase, it was verified that the ethanolic extract from seeds of A. mucosa (AMSE) was the most promising one, causing significant acute toxicity (LC50 and LC90 of 842.97 and 1,822.00 mg kg-1, respectively) and pronounced inhibition of larval development (chronic toxicity) after a 7-day exposition to treated diets. At LC50, AMSE decreased larval and pupal viabilities and also the pupal weight at 24 hours, and increased the larval phase duration. However, AMSE didn\'t cause antifeedant effect for 4-instar S. frugiperda larvae, though it decreased consumption throughout the time. After that, AMSE efficacy was compared to commercial pesticides from natural (Azamax® 1.2 EC) and synthetic (Premio® SC) origin. In this confrontation, AMSE (at LC90) showed efficacy similar to that of commercial products (used at registered dose for S. frugiperda control). Then, the bioguided fractionation using different chromatographic techniques which led to isolation of acetogenin rolliniastatin-1 identified as the majoritary compound of the most active ethanolic extract from A. mucosa. After that, rolliniastatin-1 was again tested against S. frugiperda larvae and caused significant acute (larval mortality) and chronic (decrease of larval development) effects. Finally, the bioactivity of a newly-registered acetogenin-based (Anosom® 1 EC) formulation was investigated, both separately and mixed with a limonoid-based formulation (Azamax® 1.2 EC). Anosom® 1 EC, tested at estimated LC90 (2,959.00 mg kg-1) and Azamax® 1.2 EC, tested at registered dose for S. frugiperda (4,000.00 mg kg-1) led to significant larval mortality, without happening any differences between treatments either tested separately or in a binary mixture. Anosom® 1 EC (at LC50) also led to significant increase of larval and pupal mortality and larval phase, as well as insect pupal weight decrease. Thus, Annonaceae derivates can be a useful component for S. frugiperda integrated management under field conditions.

In 2004, the polyketide natural product, chamuvarinin (72) was isolated by Laurens et al. from the roots of Uvaria chamae, a member of the Annonaceae plant family. This unique tetrahydropyran containing acetogenin displayed potent levels of cytotoxic activity against the KB 3-1 cell line with an ED50 value of 0.8 nM. Upon initial isolation the relative and absolute stereochemical assignment of chamuvarinin (72) was unable to be readily achieved through ¹H and ¹³C NMR analysis. The initial synthetic route described herein has enabled the relative and absolute stereochemical determination of chamuvarinin (72) through the first total synthesis completed in 20 longest linear steps in 1.5% overall yield. A revised synthetic strategy towards chamuvarinin (72) was completed in 17 longest linear steps in 2.2% overall yield. The revised route facilitated the assembly of non-natural chamuvarinin-like analogues and their trypanocidal and cytotoxic activities have been assessed. The synthesis of these analogues has formed the basis of a more focussed study through the design and synthesis of simplified triazole (295), isoxazole (325) and butenolide triazole (305) analogues as potential Trypanosoma brucei (causative agent in African Sleeping sickness) inhibitors.

Une importante proportion de formes atypiques de parkinsonismes a été rapportée en Guadeloupe en 1999. Il ressort des études épidémiologiques menées sur place, que tous les patients atteints étaient de grands consommateurs de produits alimentaires et médicinaux de la famille des Annonaceae, et plus particulièrement du genre Annona. De nombreux genres appartenant à cette famille renferment des molécules fortement cytotoxiques : les acétogénines d’Annonaceae. Leur présence dans les fruits d’Annona muricata a déjà été mise en évidence. Cependant, peu de données existent sur leur teneur en acétogénines dans ces fruits ou dans ceux d’espèces proches. Par ailleurs, bien que l’annonacine, acétogénine principale d’A. muricata soit neurotoxique in vivo, aucune donnée quantitative de son accès au cerveau n’est disponible. Nous nous sommes donc attachés au cours de ce travail, à développer des méthodes de dosage de l’annonacine par LC-MS/MS dans le plasma et le cerveau de Rat pour déterminer sa biodisponibilité et la fraction à tropisme cérébral, dans le but de comprendre pourquoi des molécules si fortement cytotoxiques n’entraînaient pas d’intoxication aigue lors de l’exposition alimentaire. Nous avons par ailleurs développé une méthode de quantification des acétogénines totales par 1H RMN dans des extraits bruts de fruits, appliquée à des lots d’A. muricata d’origines variées. Une méthode par LC-MS/MS a également été développée pour une description plus approfondie des acétogénines présentes dans des extraits bruts de fruits, appliquée à différents lots d’A. squamosa. Les fruits d’A. reticulata et d’A. glabra ont également fait l’objet d’investigations. Ces deux approches combinées ont contribué à améliorer l’estimation de l’exposition aux acétogénines dans le cadre de l’alimentation. / Abstract : A high proportion of atypical parkinsonisms was reported in French West Indies in 1999. Epidemiological studies pointed out an association with the consumption of fruits and medicinal herbs from Annonaceae of the Annona genera. Numerous Annonaceae members contain Annonaceous acetogenins (AAGs), which are highly cytotoxic molecules. They were found in the pulp fruit of Annona muricata. However, scarce only quantitative exist for this fruit and those of related species. Moreover, although annonacin, the major AAG of A. muricata proved neurotoxic in vivo, no quantitative data is available towards its distribution to the brain. We therefore developed a method for annonacin quantitation in Rat plasma and brain homogenate, in order to determine its bioavailability and the fraction reaching the brain, to understand why those highly cytotoxic molecules are not responsible for acute toxicity when fruits are ingested. We then developed a quantitation method for global estimation of AAGs in crude fruit extracts by 1H NMR, which we applied to the fruit pulp of A. muricata batches from diverse locations. An LC-MS/MS method was also developed for the qualitative study of AAGs. It was applied to different batches from A. squamosa fruits. The species A. reticulata and A. glabra were also examined. Those two approaches contributed in a better estimation of AAGs exposure by fruit consumption.

Dans les Antilles françaises, une proportion anormalement élevée de parkinsonismes atypiques sporadiques – des tauopathies – est observée. Un lien avec la consommation de plantes de la famille des Annonaceae, en particulier Annona muricata L. (corossol) a été démontré. Les acétogénines d’Annonaceae, des inhibiteurs puissants du complexe I de la chaine respiratoire mitochondriale, sont considérées comme des toxines candidates. L’annonacine, une acétogénine représentative, majoritaire dans A. muricata, est neurotoxique in vitro et in vivo. L’Agence Française de Sécurité Sanitaire des Aliments a exprimé ses doutes quant à ce problème de santé publique. Elle insiste sur l’importance d’évaluer l’exposition des consommateurs d’Annonaceae aux acétogénines, et de déterminer les paramètres pharmacocinétiques de ces molécules. Au cours de cette thèse, nous avons cherché à répondre à ces interrogations, avec l’annonacine pour modèle. Après l’analyse structurale d’acétogénines étalons, une méthode de déréplication puissante et innovante a été mise au point par CLHP-ESI-LTQ-Orbitrap® avec infusion post-colonne de lithium. Les profils complets des acétogénines d’extraits bruts issus d’un nectar d’A. muricata et d’un alcool d’Annona cherimolia Mill. (annone, chérimole) ont été élucidés, mettant en évidence une composition plus complexe et plus variée que celle envisagée dans la littérature. A. cherimolia n’avait pas été identifiée comme une source d’exposition jusqu’à maintenant. Des données quantitatives ont été obtenues par CLHP-DAD-MS, à partir d’une quinzaine d’échantillons de produits commerciaux, confirmant une exposition humaine importante à ces molécules par voie alimentaire, via des produits d’origines géographiques, de statuts et de modes d’obtention variés. Des travaux préliminaires d’étude du passage de l’annonacine à travers des membranes biologiques ont été amorcés (modèles de barrières intestinale – Caco-2 – et hémato-encéphalique – hCMEC/D3). Une étude de métabolisation de phase I de l’annonacine sur microsomes de foie de Rat a permis d'identifier 25 métabolites mono-hydroxylés par CLHP-ESI-LTQ-Orbitrap®. Seuls trois d’entre eux sont observés avec des microsomes humains. Ces métabolites ont été obtenus par hémisynthèse (bioconversion, catalyse par porphyrines) et leur structure a été déterminée. Les résultats montrent que cette étape de métabolisation n’est pas cruciale dans le devenir de l’annonacine, et ne peut expliquer de susceptibilité différentielle aux acétogénines. Après la présentation de rappels concernant les Annonaceae, les parkinsonismes et leurs formes atypiques guadeloupéennes et tropicales, puis d’aspects méthodologiques en spectrométrie de masse, nos travaux de phytochimie analytique, d’analyse métabolique, d’hémisynthèse et de détermination structurale sont présentés, et discutés en regard d’un problème de santé publique potentiellement large et préoccupant. / In the French West Indies, an unusually high proportion of atypical sporadic parkinsonisms - tauopathies - is observed. A link between these atypical parkinsonisms and the consumption of plants of the Annonacea family, Annona muricata L. (soursop) was demonstrated. The Annonaceous acetogenins are potent inhibitors of complex I of mitochondrial respiratory chain and are considered to be in vitro toxins candidate. The major acetogenin in Annona muricata, annonacin, is neurotoxic in in vitro and in vivo models. Afssa (Agence Française de Sécurité Sanitaire des Aliments) expressed its concern regarding this public health problem. The Agency reports the importance of assessing consumers ‘exposure to Annonaceous acetogenins, and of determining the pharmacokinetic of these molecules. In this thesis, we sintended to answer these questions, with annonacine as a model. After the structural analysis of acetogenins standards, a powerful and innovative method of dereplication was developed by HPLC-ESI-LTQ-Orbitrap ® with post-column infusion of lithium. Complete acetogenins profile in crude extracts from nectar of A. muricata and from an alcohol of Annona cherimolia Mill. (Annona, cherimoya) were elucidated, revealing a more complex and more varied composition than that proposed in the literature. A. cherimolia had not been identified as a source of exposure to date. Quantitative data were obtained by HPLC-DAD-MS, from fifteen samples of commercial products, confirming an important human exposure to these molecules through food products of varied geographical origins, status and methods. Preliminary works to study ability of annonacin to cross biological membranes have been initiated (intestinal barrier models - Caco-2 - and blood-brain barrier - hCMEC/D3). A study of phase I metabolism of annonacin in rat liver microsomes allowed the identification of 25 mono-hydroxylated metabolites by HPLC-ESI-LTQ-Orbitrap®. Only three of them were observed with human microsomes. These metabolites were obtained by semisynthesis (bioconversion, porphyrin mediated catalysis) and their structure was determined. The results show that phase metabolism is not critical in the becoming of annonacin, and cannot explain a differential susceptibility of acetogenins. After a presentation of the Annonaceae family, of parkinsonism – including atypical and guadeloupean forms then of and methodological aspects of mass spectrometry, analytical phytochemistry of our work, metabolic analysis, semisynthesis and structure determination are proposed, and discussed in the context of a public health problem and potentially broad concern.