Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Student: Lenka Srbová Supervisor: PharmDr. Marie Kašparová, Ph.D. Title of diploma thesis: Tissue culture of Juniperus virginiana L. as a promising source of podophyllotoxin. The diploma thesis deals with the cultivation of Juniperus virginiana tissue cultures. A growth was observed at Juniperus virginiana two years old culture (variety 'Glauca' and 'Grey Owl') after adding various concentrations of phenylalanine (1 mmol.l-1, 10 mmol.l-1 and 100 mmol.l-1 ) at selected time intervals. The results show that the highest increase in callus fresh weight was detected at Juniperus virginiana variety 'Grey Owl', particularly on the 14th day after adding 10 mmol.l-1 phenylalanine. Suspension culture was successfully derived from the Juniperus virginiana two years old callus culture (variety 'Glauca').

É notório o potencial farmacológico de lignanas, metabólitos secundários extraídos de produtos naturais, na sua maioria com baixos rendimentos. Usando a natureza como fonte de inspiração, foram propostas rotas sintéticas de compostos análogos, obtendo-se o 2,5-bis(2-metoxifenil)tiofeno, um análogo químico da lignana grandisina. De acordo com o potencial leishmanicida preliminarmente identificado, associado à reduzida citotoxicidade do análogo, despertou-se o interesse para o desenvolvimento de nova terapia medicamentosa para as leishmanioses em substituição aos tratamentos rotineiros e tóxicos utilizados. Entretanto, não há na literatura informações sobre os processos que abrangem a farmacocinética - absorção, distribuição, metabolismo e excreção (ADME) do composto. Portanto, o presente estudo objetivou a avaliação pré-clínica da disposição cinética do 2,5-bis(2-metoxifenil)tiofeno. Inicialmente foi desenvolvido um método analítico por cromatografia liquida de ultra eficiência acoplada a espectrometria de massas (CLUE-EM/EM) para quantificação do analito extraído da matriz de plasma, através de precipitação de proteínas com acetonitrila acidificada. O método foi validado de acordo com guias oficiais da Agência Nacional de Vigilância Sanitária e da Food and Drug Adminstration, mostrando-se sensível, exato, preciso e linear na faixa de 4 a 1000 ng.mL-1. A partir da administração intravenosa do 2,5-bis(2-metoxifenil)tiofeno em ratos foi possível obter curvas de decaimento plasmático versus tempo, observando-se que os dados adequaram-se ao modelo bicompartimental. O perfil farmacocinético do 2,5-bis(2-metoxifenil)tiofeno indicou rápida distribuição (t1/2? de 10,66 ± 2,37 min) e eliminação (t1/2? de 71,68 ± 13,84 min), não provocando acúmulo plasmático. / It is notorious the pharmacological potential of lignans, secondary metabolites extracted from natural products, mostly with low yields. Applying nature as a source of inspiration, synthetic routes of analogous compounds have been proposed getting 2,5-bis(2-methoxyphenyl)thiophene, a chemical analogue of lignan grandisine. According to the leishmanicide potential preliminarily identified, associated with the reduced cytotoxicity of the analogue, interest was aroused for the development of new drug therapy for leishmaniasis, replacing the typical and toxic treatments used. However, there is no information about the pharmacokinetics processes - absorption, distribution, metabolism and excretion (ADME) of the compound in the literature. Therefore, the present study aimed to evaluate the kinetic disposition of 2,5-bis (2-methoxyphenyl) thiophene. An analytical method was developed by ultra performance liquid chromatography coupled to mass spectrometry (UPLC-MS/MS) for quantification of the analyte extracted from the plasma matrix through precipitation of proteins with acidified acetonitrile. The method was validated in according to Agência Nacional de Vigilância Sanitária and Food and Drug Adminstration guidelines, showing to be sensitive, accurate, precise and linear over the concentration range of 4 to 1000 ng.mL-1. From the intravenous administration of 2,5-bis(2-methoxyphenyl)thiophene in rats it was possible to obtain the mean plasma concentration versus time profile, and the concentration data was best fitted to a two-compartment model. The pharmacokinetic profile of 2,5-bis (2-methoxyphenyl) thiophene indicated rapid distribution (t1 / 2? of 10.66 ± 2.37 min) and elimination (t1 / 2? of 71.68 ± 13.84 min), without plasma accumulation.

Neste trabalho o metilpluviatolido (1), o matairesinol (7), a hinoquinina (11), a cubebina (17) e seus respectivos derivados, providos de algumas modificações estruturais foram submetidos a testes in vitro para a determinação da atividade citotoxica e tripanocida. As substâncias não apresentaram atividade citotoxica significativa para a linhagem celular utilizada (LLC-MK2). Nos ensaios tripanocida in vitro o dimetoximorelensin (8) e 11 foram as substâncias que determinaram as maiores atividades tripanocida sobre as varias formas e cepas de T. cruzi utilizadas e juntamente com a cubebina foram submetidas os ensaios in vivo. As substâncias determinaram alterações significativas no curso da infecção chagásica experimental. No entanto apenas a 8 apresentou efeito semelhante ao do benzonidazol para ambas as cepas, sendo a cepa BOL em relação a cepa Y mais resistente as substâncias 11 e 17. Algumas rotas metabólicas foram avaliadas como mecanismo de ação das substâncias. As substâncias não apresentaram atividade inibitória sobre a enzima gliceraldeído-3-fosfato desidrogenase (GAPDH) de Trypanosoma cruzi, não induziram a produção de H2O2 e outros peróxidos, bem como não induziram a produção de óxido nítrico, tendo algumas inibindo esta produção. No entanto, apresentaram efeito inibitório sobre a respiração celular e efeito inibitório sobre a enzima ferro superóxido dismutase (Fe-SOD) de Trypanosoma cruzi. Adicionalmente foi demonstrado que em sistema livre de células as substâncias não apresentam atividade scavenger de radicais livres e que o tratamento com a substancia 8, avaliado por microscopia eletrônica, provoca alterações nucleares nas formas epimastigotas do parasita, sugerindo que a atividade tripanocida desta substancia envolva a inibição da síntese de DNA e RNA. Dessa forma, podemos concluir que a atividade tripanocida das varias lignano-lactonas avaliadas é promissora e que a produção de H2O2 e outros peróxidos, atividade inibitória sobre a enzima gliceraldeído-3-fosfato desidrogenase (GAPDH) de Trypanosoma cruzi, bem como a capacidade de indução de óxido nítrico pelos derivados bioativos são mecanismos que parecem não estar envolvidos na atividade tripanocida das substâncias. Além disso, que o efeito inibitório sobre a respiração celular parece contribuir para a atividade tripanocida e pode estar envolvido no mecanismo de citotoxicidade destas substâncias. Ainda, que o efeito inibitório sobre a enzima ferro superóxido dismutase (Fe- SOD) de Trypanosoma cruzi parece ser um dos principais mecanismos envolvidos na atividade tripanocida das substâncias. / In this work the compounds methylpluviatolide (1), matairesinol (7), hinokinin (11), cubebin (17) and its derivatives bearing some structural modifications were submitted to in vitro trypanocidal and citotoxic assays. The compounds do not show significant citotoxicity for the LLC-MK2 cells used. In the in vitro trypanocidal assays, the compounds more active against the different strains and forms of T. cruzi were the dimethoxymorelensin (8) and hinokinin (11). These compounds and cubebin (17) were submitted to in vivo trypanocidal assay. The compounds display significant modifications on the experimental chagasic infection. However, for both strains, Y and BOL, only the 8 displayed similar effect of the benznidazole. The BOL strain was more resistant than Y strain to treatment with 11 and 17. Some metabolic routes were evaluated as compounds action mechanism. The compounds do not showed inhibitory activity under enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Trypanosoma cruzi, they do not induced the production of H2O2 and others peroxides and also do not induced the nitric oxide production, instead some compounds decrease this production. On the other hand, they showed inhibitory effect under the cellular respiration and the enzyme iron superoxide dismutase (Fe-SOD) of Trypanosoma cruzi. In addition, it was demonstrated that on system cells free, the compounds do not showed free radicals scavenger activity and that the treatment with the compound 8, evaluated in electron microscopy, display nuclear alterations on epimastigotes forms, suggesting an inhibitory effect on T. cruzi DNA and RNA biosynthesis. In conclusion, the compounds trypanocidal activity is promising and the production of H2O2 and others peroxides, the activity under enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Trypanosoma cruzi, and the induction of nitric oxide production seem do not be involved in the trypanocidal and citotoxity activity of these group of compounds. The inhibitory effect under cellular respiration seems to be contributing for trypanocidal activity and may be involved on its citotoxic mechanism. Moreover, the inhibitory effect under the enzyme iron superoxide dismutase (Fe-SOD) of Trypanosoma cruzi seems to be the main mechanism involved on trypanocidal activity of these compounds.

In order to investigate the structural features responsible for the cytotoxicity of the naturally occurring lignan nordihydroguaiaretic acid, the synthesis of four structural analogues of NDGA is proposed for the purpose of studying their oxidative metabolism. One analogue in particular (1), a mono-catechol analogue, is successfully synthesized employing a double Stobbe condensation approach. Following synthesis of this compound a series of oxidation experiments is performed consisting of: incubation in rat liver microsomes with and without the trapping agent glutathione (GSH), oxidation with mushroom tyrosinase, oxidation with silver oxide, and oxidation with horseradish peroxidase. Results are analyzed via HPLC and UPLC-MS. It is found that 1 does not autoxidize at pH 7.4 as NDGA does. Two products are produced during incubation of 1 in rat liver microsomes with UPLC-ESI(-)-MS results giving m/z of 879.2 and 574.18. This is consistent with 1 plus 2 GSH and 1 plus 1 GSH respectively; confirming 1 will oxidize to an electrophilic moiety. Oxidation with mushroom tyrosinase is found to produce high levels of product two with m/z 574.2. Oxidation with horseradish peroxidase is found to produce high levels of the m/z 879.2 product. Silver Oxide produced multiple products rather than the expected one major product, but most are found to be inconsistent with the products seen during rat liver microsomal incubation, and are not pursued.

In order to investigate the structural features responsible for the cytotoxicity of the naturally occurring lignan nordihydroguaiaretic acid, the synthesis of four structural analogues of NDGA is proposed for the purpose of studying their oxidative metabolism. One analogue in particular (1), a mono-catechol analogue, is successfully synthesized employing a double Stobbe condensation approach. Following synthesis of this compound a series of oxidation experiments is performed consisting of: incubation in rat liver microsomes with and without the trapping agent glutathione (GSH), oxidation with mushroom tyrosinase, oxidation with silver oxide, and oxidation with horseradish peroxidase. Results are analyzed via HPLC and UPLC-MS. It is found that 1 does not autoxidize at pH 7.4 as NDGA does. Two products are produced during incubation of 1 in rat liver microsomes with UPLC-ESI(-)-MS results giving m/z of 879.2 and 574.18. This is consistent with 1 plus 2 GSH and 1 plus 1 GSH respectively; confirming 1 will oxidize to an electrophilic moiety. Oxidation with mushroom tyrosinase is found to produce high levels of product two with m/z 574.2. Oxidation with horseradish peroxidase is found to produce high levels of the m/z 879.2 product. Silver Oxide produced multiple products rather than the expected one major product, but most are found to be inconsistent with the products seen during rat liver microsomal incubation, and are not pursued.

Neste trabalho o metilpluviatolido (1), o matairesinol (7), a hinoquinina (11), a cubebina (17) e seus respectivos derivados, providos de algumas modificações estruturais foram submetidos a testes in vitro para a determinação da atividade citotoxica e tripanocida. As substâncias não apresentaram atividade citotoxica significativa para a linhagem celular utilizada (LLC-MK2). Nos ensaios tripanocida in vitro o dimetoximorelensin (8) e 11 foram as substâncias que determinaram as maiores atividades tripanocida sobre as varias formas e cepas de T. cruzi utilizadas e juntamente com a cubebina foram submetidas os ensaios in vivo. As substâncias determinaram alterações significativas no curso da infecção chagásica experimental. No entanto apenas a 8 apresentou efeito semelhante ao do benzonidazol para ambas as cepas, sendo a cepa BOL em relação a cepa Y mais resistente as substâncias 11 e 17. Algumas rotas metabólicas foram avaliadas como mecanismo de ação das substâncias. As substâncias não apresentaram atividade inibitória sobre a enzima gliceraldeído-3-fosfato desidrogenase (GAPDH) de Trypanosoma cruzi, não induziram a produção de H2O2 e outros peróxidos, bem como não induziram a produção de óxido nítrico, tendo algumas inibindo esta produção. No entanto, apresentaram efeito inibitório sobre a respiração celular e efeito inibitório sobre a enzima ferro superóxido dismutase (Fe-SOD) de Trypanosoma cruzi. Adicionalmente foi demonstrado que em sistema livre de células as substâncias não apresentam atividade scavenger de radicais livres e que o tratamento com a substancia 8, avaliado por microscopia eletrônica, provoca alterações nucleares nas formas epimastigotas do parasita, sugerindo que a atividade tripanocida desta substancia envolva a inibição da síntese de DNA e RNA. Dessa forma, podemos concluir que a atividade tripanocida das varias lignano-lactonas avaliadas é promissora e que a produção de H2O2 e outros peróxidos, atividade inibitória sobre a enzima gliceraldeído-3-fosfato desidrogenase (GAPDH) de Trypanosoma cruzi, bem como a capacidade de indução de óxido nítrico pelos derivados bioativos são mecanismos que parecem não estar envolvidos na atividade tripanocida das substâncias. Além disso, que o efeito inibitório sobre a respiração celular parece contribuir para a atividade tripanocida e pode estar envolvido no mecanismo de citotoxicidade destas substâncias. Ainda, que o efeito inibitório sobre a enzima ferro superóxido dismutase (Fe- SOD) de Trypanosoma cruzi parece ser um dos principais mecanismos envolvidos na atividade tripanocida das substâncias. / In this work the compounds methylpluviatolide (1), matairesinol (7), hinokinin (11), cubebin (17) and its derivatives bearing some structural modifications were submitted to in vitro trypanocidal and citotoxic assays. The compounds do not show significant citotoxicity for the LLC-MK2 cells used. In the in vitro trypanocidal assays, the compounds more active against the different strains and forms of T. cruzi were the dimethoxymorelensin (8) and hinokinin (11). These compounds and cubebin (17) were submitted to in vivo trypanocidal assay. The compounds display significant modifications on the experimental chagasic infection. However, for both strains, Y and BOL, only the 8 displayed similar effect of the benznidazole. The BOL strain was more resistant than Y strain to treatment with 11 and 17. Some metabolic routes were evaluated as compounds action mechanism. The compounds do not showed inhibitory activity under enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Trypanosoma cruzi, they do not induced the production of H2O2 and others peroxides and also do not induced the nitric oxide production, instead some compounds decrease this production. On the other hand, they showed inhibitory effect under the cellular respiration and the enzyme iron superoxide dismutase (Fe-SOD) of Trypanosoma cruzi. In addition, it was demonstrated that on system cells free, the compounds do not showed free radicals scavenger activity and that the treatment with the compound 8, evaluated in electron microscopy, display nuclear alterations on epimastigotes forms, suggesting an inhibitory effect on T. cruzi DNA and RNA biosynthesis. In conclusion, the compounds trypanocidal activity is promising and the production of H2O2 and others peroxides, the activity under enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Trypanosoma cruzi, and the induction of nitric oxide production seem do not be involved in the trypanocidal and citotoxity activity of these group of compounds. The inhibitory effect under cellular respiration seems to be contributing for trypanocidal activity and may be involved on its citotoxic mechanism. Moreover, the inhibitory effect under the enzyme iron superoxide dismutase (Fe-SOD) of Trypanosoma cruzi seems to be the main mechanism involved on trypanocidal activity of these compounds.

No description available.

The intramolecular styryl Diels – Alder (ISDA) reaction is a rare and unique [4+2] cycloaddition with potential in the syntheses of polycycles. Its utility is based on the formation of two rings and one stereocenter in a single step, making it an efficient method for the construction of lignan-type natural product targets. Detailed mechanistic studies with complex esters and the application to natural product synthesis has been limited due to drawbacks including the loss of aromaticity, producing slow reactivity, a potentially problematic thermal [1,3]-hydrogen shift, and electronic mismatch related to the substituents on the aryl functional groups. In this research, we found conditions that led to the successful application of the ISDA reaction on the total synthesis of the anticancer deoxypodophyllotoxin and the antiviral agent F4-4. Deoxypodophyllotoxin was synthesized in seven steps, which is a very concise synthesis for a complex lignan. Density functional theory was used to analyze the two components of the ISDA reaction, the [4+2] cycloaddition and the [1,3]-hydrogen shift. Several pathways were analyzed, and the rate determining step was determined to be the [4+2] cycloaddition. We also found that the [1,3]-hydrogen shift is assisted by di-tert-butylhydroxytoluene and is lower in energy than the [4+2] cycloaddition.The two targets chosen for this research have important biological activities. Deoxypodophyllotoxin is known as a potent anticancer agent related to podophyllotoxin. Podophyllotoxin is a more abundant lignan which is the precursor of the FDA approved drugs etoposide and teniposide, used for the treatment of lung and testicular cancer. Other biological activities of deoxypodophyllotoxin have been found including antibacterial, antiviral, and anti-inflamatory activity. Also, it was recently discovered that F4-4 possesses antiviral activities against Herpes simplex viruses 1 (HSV-1), 2(HSV-2), and H. zoster. Since both deoxypodophyllotoxin and F4-4 are not available in large quantities from natural sources, chemical synthesis is important for continuing research and drug development of these compounds.

As part of an International Cooperative Biodiversity Group (ICBG) program and a continuing search for antiproliferative natural products from the Madagascar rainforest, and a collaborative research project established between Virginia Tech and the Institute for Hepatitis and Virus Research (IHVR) focusing on searching for bioactive natural products from tropical forests in South Africa, 20 extracts were selected for investigation based on their antiproliferative activities against A2780 human ovarian cancer cell line or antimalarial activities against the Dd2 strain of Plasmodium falciparum. Bioassay-guided fractionation of seven of the extracts yielded twenty new compounds and twenty-four known compounds, and their structures were elucidated by using a combination of 1D (1H and 13C) and 2D NMR spectroscopy including COSY, HASQC, HMQC, HMBC, and NOESY sequences, mass spectrometry, UV, IR, ECD, optical rotation, and chemical conversions. In addition, ten known compounds were isolated from another five of the extracts, while studies on the remaining extracts were suspended due to loss of activity, unworkable small amounts of material, or low structural interest. The plants and their metabolites are discussed in the following order: five new antimalarial 5,6-dihydro-𝛼-pyrones and six bicyclic tetrahydro-𝛼-pyrone derivatives from Cryptocarya rigidifolia (Lauraceae); two new and five known antiproliferative lignans from Cleistanthus boivinianus (Phyllanthaceae); two new and two known antiproliferative sesquiterpenes lactones from Piptocoma antillana (Asteraceae); one new antiproliferative 1,4-naphthoquinone, one known antiproliferative isoflovonoid, and five known antiproliferative stilbenoids from Stuhlmannia moavi (Leguminosae); four known antiproliferative bisbenzylisoquinoline alkaloids from Anisocycla grandidieri (Menispermaceae); one new and two known antiproliferative butanolides, and two new antiproliferative secobutanolides from Ocotea macrocarpa (Lauraceae); one new antiproliferative and five known antiproliferative diterpenoids from Malleastrum rakotozafyi (Meliceae); and 10 known compounds from Monoporus sp. (Myrsinaceae), Premna corymbosa (Verbenaceae), Premna perplexanes (Verbenaceae), Epallage longipes (Asteraceae), and Cinnamosma fragrans (Canellaceae). / Ph. D.

Made available in DSpace on 2015-05-14T13:00:03Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2069742 bytes, checksum: 30969d68a1c51eddd37190cde04e71ac (MD5) Previous issue date: 2014-06-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Leishmaniasis is a complex of inserted parasitic diseases in the group of neglected tropical diseases. The drugs used primarily in the treatment of leishmaniasis are pentavalent antimonials and amphotericin B. However, these drugs are associated with serious side effects. Therefore, it is necessary to search for new antileishmanial drugs. In this context natural products are a source of new active molecules. This study, it was investigated the antileishmanial activity of the ethanol extract of Phyllanthus acuminatus and Justicidin B, lignan obtained from this plant and the ethanol extract, the dichloromethane phase and Hyptenolide obtained from Hyptis macrostachys in vitro experimental models. All samples assessed showed antileishmanial activity, initially demonstrated by inhibition of growth of L. amazonensis promastigotes in logarithmic growth phase. The concentration that inhibits 50% of growth (IC50) for the tested substances was 35.58 μg /mL for the ethanolic extract of P. acuminatus, 12.51 μg/mL for Justicidin B; 280.69 μg/mL for the ethanol extract of H. macrostachys, 55.3 μg/mL for dichloromethane phase of H. macrostachys and 22.67 μg/mL for Hyptenolide. These samples were also evaluated for cytotoxicity in murine peritoneal macrophages of swiss mice, which resulted in the measurement of the cytotoxic concentration for 50% macrophages (CC50), which were 39.62 μg/mL for the ethanolic extract of P. acuminatus, 58.56 μg/ml for Justicidin B; 121,36 μg/mL for the ethanol extract of H. macrostachys, 40.45 μg/mL for dichloromethane phase of H. macrostachys and 61.78 μg/ml for Hyptenolide. These data resulted in IC50 and CC50 resulted in a selectivity index of 1.11 for the ethanol extract of P. acuminatus; 4.68 for Justicidin B; 0.43 for the ethanol extract of H. macrostachys; 0.73 to dichloromethane phase of H. macrostachys and 2.72 to Hyptenolide. Given these results, we selected the lignan Justicidina B for further study of antileishmanial activity against L. amazonensis. In the evaluation of the DNA fragmentation pattern of L. amazonensis promastigotes, it was observed in higher concentration evaluated of Justicidin B (4x IC50) induction of DNA fragmentation similar to that observed in the positive control H2O2 (4mM). In infection model macrophage with L. amazonensis and treated with lignan Justicidin B showed reduction in both the percentage of infected macrophages as the number of amastigotes per infected macrophage, resulting in a reduction in the rate of infection compared to control, resulting decrease in the effective concentration for 50% of infection (EC50) of 9.14 and 3.58 for 24 hours and 72 hours after treatment. This result was correlated with an immunomodulatory activity of this lignan, associated with decreased levels of interleukin (IL) -10, and increased levels of nitric oxide (NO). It can be concluded that the lignan Justicidin B has significant activity antipromastigote and antiamastigote and presents immunomodulatory properties in models of infection in vitro. / As leishmanioses são um complexo de doenças parasitárias inseridas no grupo de doenças tropicais negligenciadas. Os principais medicamentos utilizados na terapêutica das leishmanioses são antimoniais pentavalentes e anfotericina B. Contudo, esses medicamentos estão associados a sérios efeitos colaterais. Portanto, é necessária a busca de novas drogas leishmanicidas. Nesse contexto, os produtos naturais são uma fonte de novas moléculas ativas. Neste trabalho, investigou-se a atividade antileishmania do extrato etanólico de Phyllanthus acuminatus e Justicidina B, lignana obtida dessa planta, bem como o extrato etanólico, a fase diclorometano e o Hiptenolideo, obtidos de Hyptis macrostachys em modelos experimentais in vitro. Todas as amostras avaliadas apresentaram atividade antileishmania, demonstrada inicialmente pela inibição de crescimento de formas promastigotas de L. amazonensis em fase logarítmica de crescimento. A concentração que inibe 50% do crescimento (CI50) para as substâncias avaliadas foi de 35,58μg/mL para o extrato etanólico de P. acuminatus, 12,51μg/mL para Justicidina B; 280,69μg/mL para o extrato etanólico de H. macrostachys, 55,3μg/mL para fase diclorometano de H. macrostachys e 22,67μg/mL para o Hiptenolideo. Essas amostras também foram avaliadas quanto à citotoxicidade em macrófagos peritoneais murinos de camundongos suíços, o que resultou na medida da concentração citotóxica para 50% dos macrófagos (CC50), que foram de 39,62μg/mL para o extrato etanólico de P. acuminatus, 58,56μg/mL para Justicidina B; 121,36μg/mL para o extrato etanólico de H. macrostachys, 40,45μg/mL para fase diclorometano de H. macrostachys e 61,78μg/mL para o Hiptenolideo. Esses dados de CI50 e CC50 resultaram no Índice de Seletividade que foram de 1,11 para o extrato etanólico de P. acuminatus; 4,68 para Justicidina B; 0,43 para o extrato etanólico de H. macrostachys; 0,73 para fase diclorometano de H. macrostachys e 2,72 para o Hiptenolideo. Diante desses resultados, foi selecionada a lignana Justicidina B para aprofundar os estudos de atividade antileishmania sobre L. amazonensis. Na avaliação do padrão de fragmentação do DNA de formas promastigotas de L. amazonensis observou-se que a Justicidina B na maior concentração avaliada (4x IC50) foi capaz de induzir a fragmentação do DNA semelhante ao observado no controle positivo H2O2 (4mM). Em modelo de infecção de macrófagos com L. amazonensis e tratados com a lignana Justicidina B observou-se redução tanto da porcentagem de macrófagos infectados quanto do número de amastigotas por macrófago infectado, resultando em uma redução no índice de infecção quando comparados ao controle, resultando em concentração efetiva para diminuir 50% da infecção (EC50) de 9,14 e 3,58 para 24 horas e 72 horas de tratamento. Esse resultado foi correlacionado com uma atividade imunomoduladora dessa lignana, associada à diminuição nos níveis de interleucina (IL)-10, e a um aumento nos níveis de óxido nítrico (NO). Pode-se concluir que a lignana Justicidina B possui significativa atividade antipromastigota e antiamastigota e apresenta propriedades imunomodulatórias em modelos de infecção in vitro