Made available in DSpace on 2014-12-17T14:16:30Z (GMT). No. of bitstreams: 1 AnneCOC_DISSERT.pdf: 1977864 bytes, checksum: 512fa415d2a3e86643a5d0929e22bccf (MD5) Previous issue date: 2012-06-05 / Kalanchoe brasiliensis Cambess (Crassulaceae), commonly known as sai?o , coirama branca , folha grossa , is originally from Brazil and commonly found in S?o Paulo to Bahia, mainly in the coastal zone. Regarding of biological activities, most preclinical studies were found in the literature, mainly about the anti-inflammatory activity of extracts obtained from leaves and / or aerial parts of K. brasiliensis. As regards the chemical constitution, it has been reported mainly the presence of flavonoids in the leaves of the species, but until this moment did not knows which are the active compounds. Although it is a species widely used in traditional medicine in Brazil, there is no monograph about the quality parameters of the plant drug. In this context, this study aims to characterize and quantify the chemical markers of hydroethanolic extract (HE) from the leaves of K. brasiliensis, which can be used in quality control of plant drug and derivatives obtained from this species. The methodology was divided into two parts: i. Phytochemical study: to fractionate, isolate and characterizate of the chemical (s) marker (s) of the HE from the leaves of K. brasiliensis; ii. To Developed validate of analytical method by High Performance Liquid Chromatography (HPLC)-diode array detector (DAD) to quantify the chemical (s) marker (s) of the EH. i. The EH 50% was prepared by turbo extraction method. It was then submitted to liquid-liquid partition, obtaining dichloromethane, n-butanol and ethyl acetate (AcOEt) fractions. The AcOEt fraction was selected to continue the fractionation process, because it has a chemical profile rich in flavonoids. The acOEt fraction was submitted to column chromatography using different systems for obtaining the compound Kb1. To identify this compound, it was submitted to UV analysis ii. For quantitative analysis, the EH was analyzed by HPLC, using different methods. After selecting the most appropriate method, which showed satisfactory resolution and symmetrical peaks, it was validated according to parameters in the RE 899/2003. As result, it was obtained from the AcOEt fraction the compound Kb1 (2.7 mg). Until this moment, the basic nucleus was characterized by UV analysis using shift reagents. The partial chemical structure of the compound Kb1 was identified as a flavonol, containing hydroxyls in 3 , 4 position (ring A), 5 and 7 free (ring B) and a replacement of the C3 hydroxyl by a sugar. As the analysis were performed in the HPLC coupled to a DAD, we observed that the UV spectrum of the major peaks of EH from K. brasiliensis shown similar UV spectrum. According to the literature, it has been reported the presence of patuletin glycosydes derivatives in the leaves of this species. Therefore, it is suggested that the compound Kb1 is glycosylated patuletin derivative. Probably the sugar (s) unit(s) are linked in the C3 in the C ring. . Regarding the development of HPLC analytical method, the system used consists of phase A: water: formic acid (99,7:0,3, v / v) and phase B: methanol: formic acid (99,7:0,3, v / v), elution gradient of 40% B - 58% B in 50 minutes, ccolumn (Hichrom ?) C18 (250x4, 0 mm, 5 ?m), flow rate 0.8 mL / min, UV detection at 370 nm, temperature 25 ? C. In the analysis performed with the co-injection of thecompound Kb1 + HE of K. brasiliensis was observed that it is one of the major compounds with a retention time of 12.47 minutes and had a content of 15.3% in EH of leaves from K. brasiliensis. The method proved to be linear, precise, accurate and reproducible. According to these results, it was observed that compound Kb1 can be used as a chemical marker of EH from leaves of K. brasiliensis, to assist in quality control of drug plant and its derivatives / Kalanchoe brasiliensis Cambess (Crassulaceae), conhecida popularmente como sai?o, coirama branca e folha grossa, ? origin?ria do Brasil e comumente encontrada de S?o Paulo at? a Bahia, principalmente na zona litor?nea. Quanto ? atividade microbiol?gica, estudos pr?-cl?nicos foram realizados no sentido de investigar a atividade antiinflamat?ria, para os extratos obtidos a partir folhas e/ou partes ?reas de K. brasiliensis. Em rela??o ? constitui??o qu?mica, h? relatos principalmente da presen?a de flavonoides nas folhas da esp?cie. Embora seja uma esp?cie utilizada amplamente na medicina tradicional no Brasil, n?o h? monografia que especifique os par?metros de qualidade da droga vegetal. Dentro deste contexto, o presente trabalho tem como objetivo caracterizar e a quantificar os marcadores qu?micos do extrato hidroetan?lico (EH) das folhas de K. brasiliensis, que possam ser utilizados no controle de qualidade de derivados obtidos a partir dessa esp?cie. A metodologia do estudo foi dividida em duas partes: i. Estudo fitoqu?mico: fracionamento, isolamento e caracteriza??o de marcador(es) qu?mico(s) do extrato hidroetan?lico das folhas de K. brasiliensis; ii.Desenvolvimento e valida??o de metodologia anal?tica por Cromatografia L?quida de Alta Efici?ncia (CLAE) para quantificar (o)s marcador(es) qu?mico(s) do EH. i. O EH 50% foi obtido pelo m?todo de turboextra??o. Em seguida foi submetido a uma parti??o l?quido-l?quido, obtendo-se as fra??es diclorometano, n-butanol e acetato de etila (FAcOEt). A fra??o acetato de etila foi selecionada para dar continuidade ao fracionamento, e por apresentar um perfil qu?mico rico em flavonoides, uma vez que esses metab?litos parecem estar relacionados ? atividade farmacol?gica. ii. Para a an?lise quantitativa, o EH foi analisado por CLAE, utilizando diferentes m?todos. Ap?s selecionado o m?todo mais adequado, que apresentou picos sim?tricos e resolu??o satisfat?ria, foi realizada a valida??o segundo par?metros da RE No 899/2003. Como resultados, foi obtido a partir da fra??o acetato de etila o composto Kb1 (2,7mg). Neste composto, foi caracterizado o n?cleo b?sico flavono?dico por an?lise por UV utilizando reagentes de deslocamento. O composto Kb1 foi identificado como um flavonol, com hidroxilas em 3 , 4 (anel B) e 5 e 7 livres (anel A), al?m de uma hidroxila substitu?da em C3. Como foi utilizado a CLAE acoplada a um detector de arranjo de diodos, foi poss?vel observar que os espectros UV dos picos majorit?rios do EH de K. brasiliensis s?o similares. Em estudos anteriores foi descrita a presen?a de derivados glicosilados da patuletina nas folhas dessa esp?cie. Portanto, sugere-se que Kb1 seja um derivado glicosilado na posi??o C-3 da patuletina. Quanto ao desenvolvimento anal?tico por CLAE, o sistema utilizado foi constitu?do de fase A: ?gua:?cido f?rmico (99,7:0,3, v/v) e fase B: metanol:?cido f?rmico (99,7:0,3, v/v) em elui??o gradiente de 40%B - 58%B em 50 minutos, coluna cromatogr?fica (Hichrom) C18 (250x4,0mm, 5?m), fluxo 0,8 mL/min, detec??o em UV a 370 nm, temperatura de 25oC. Por meio de co-inje??o do padr?o + extrato foi observado que Kb1 trata-se de um dos compostos majorit?rios, com tempo de reten??o de 12,47 minutos e apresentou um teor de 15,3% no EH de K. brasiliensis O m?todo apresentou-se linear, preciso, exato e reprodut?vel. De acordo com os resultados obtidos, foi observado que Kb1 pode ser utilizado como marcador qu?mico do EH das folhas de K. brasiliensis, para auxiliar no controle de qualidade da droga vegetal e seus derivados

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2018-01-15T22:12:26Z No. of bitstreams: 1 EdilaneRodriguesDantasDeAraujo_DISSERT.pdf: 4618681 bytes, checksum: 5ed4bc7a898bcd1ef47ef38661580242 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2018-01-18T13:59:07Z (GMT) No. of bitstreams: 1 EdilaneRodriguesDantasDeAraujo_DISSERT.pdf: 4618681 bytes, checksum: 5ed4bc7a898bcd1ef47ef38661580242 (MD5) / Made available in DSpace on 2018-01-18T13:59:07Z (GMT). No. of bitstreams: 1 EdilaneRodriguesDantasDeAraujo_DISSERT.pdf: 4618681 bytes, checksum: 5ed4bc7a898bcd1ef47ef38661580242 (MD5) Previous issue date: 2017-06-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Kalanchoe brasiliensis e Kalanchoe pinnata (Crassulaceae), conhecidas como ?sai?o? e ?coirama?, t?m amplo uso popular no tratamento de ?lceras p?pticas e inflama??es cut?neas. Vale destacar que K. pinnata est? presente na Rela??o Nacional de Plantas de Interesse do Sistema ?nico de Sa?de - RENISUS (2009). Dentro deste contexto, o objetivo do presente estudo foi caracterizar os marcadores qu?micos nos sucos das folhas das duas esp?cies e avaliar as atividades gastroprotetora e anti-inflamat?ria t?pica. Foi realizada caracteriza??o fitoqu?mica por Cromatografia em Camada Delgada (CCD) e Cromatografia L?quida de Ultra Efici?ncia Acoplada a Espectr?metro de Massas (CLUE-EM). A atividade gastroprotetora foi avaliada nos modelos de ?lcera ?guda induzida por etanol e indometacina, j? a secre??o g?strica foi avaliada no modelo de ligadura do piloro, em ratos Wistar. Foi realizado o pr?- tratamento com os sucos nas doses de 125, 250 e 500 mg/kg e a ranitidina (50 mg/kg) por via oral. A atividade anti-inflamat?ria t?pica foi avaliada no modelo de edema de pata induzido por carragenina e edema de orelha induzido por ?leo de crot?n em camundongos Swiss, utilizando formula??es na forma de gel contendo os sucos em diferentes concentra??es (1,25%, 2,5% e 5%) e como f?rmaco padr?o a dexametasona (1mg/g), todos administrados por via t?pica imediatamente ap?s a indu??o. A an?lise por CCD revelou a presen?a de manchas caracter?sticas de flavonoides nos sucos das duas esp?cies, ap?s revela??o com o Reagente Natural A, sendo observado que as duas esp?cies t?m perfil flavono?dico diferente. Na an?lise por CLUE-EM K. brasiliensis apresentou flavonoides glicosilados derivados principalmente da patuletina, enquanto que K. pinnata apresentou flavonoides glicosilados derivados principalmente da quercetina. O pr?-tratamento com o suco das folhas de K. brasiliensis nas doses de 125 mg/kg (P<0,01), 250 mg/kg e 500 mg/kg (P<0,001) e K. pinnata nas doses de 125 mg/kg (P<0,01), 250 mg/k e 500 mg/kg (P<0,001) reduziram significativamente as les?es em compara??o ao controle positivo no modelo de indu??o por etanol. No modelo de indu??o por indometacina o suco das folhas de K. brasiliensis apresentou resultado significativo nas doses de 250 (P<0,05) e 500 mg/kg (P<0,01) e K. pinnata nas doses de 250 e 500 mg/kg (P<0,001). A redu??o das les?es foi acompanhada de aumento do conte?do total de glutationa e redu??o dos n?veis de malondialde?do. Al?m disso, houve redu??o dos n?veis de mieloperoxidase, IL-1? e TNF-?. Tamb?m foi observado efeito citoprotetor na avalia??o histol?gica com H&E e manuten??o da produ??o de muco com PAS, al?m da redu??o da express?o de iNOS e NF-?B p65 e aumento da express?o de ZO-1 por imunohistoqu?mica. Os sucos das folhas das duas esp?cies n?o alteraram a acidez, o pH e o volume do suco g?strico. No modelo de edema de orelha, as formula??es contendo as tr?s concentra??es do suco das folhas de K. brasiliensis reduziram significativamente o edema quando comparadas ao grupo placebo (1,25% P<0,05; 2,5% P<0,01 e 5% P<0,01). Entretanto, apenas a formula??o contendo o suco das folhas de K. pinnata na concentra??o de 5% apresentou resultado significativo (P<0,01). No modelo de edema de pata, as formula??es contendo o suco das folhas de K. brasiliensis nas concentra??es de 1,25 e 2,5% reduziram significativamente (P<0,05) o edema no tempo 4 h. A formula??o na concentra??o de 5% reduziu significativamente o edema nos tempos 1 h (P<0,001), 2 h, 3 h e 4 h (P<0,01). Em rela??o as formula??es contendo o suco das folhas de K. pinnata, a concentra??o de 1,25% reduziu significativamente o edema no tempo 1h (P<0,01) e 2h (P<0,05), na concentra??o de 5% reduziu significativamente no tempo 1h (P<0,05). A diminui??o do edema foi acompanhada da redu??o de miloperoxidase. Concluiu-se que os sucos das duas esp?cies apresentaram atividade gastroprotetora e anti-inflamat?ria t?pica em modelos in vivo, resultados que justificam a utiliza??o popular das esp?cies. / Kalanchoe brasiliensis and Kalanchoe pinnata (Crassulaceae), known as "sai?o" and "coirama", have wide popular use in the treatment of peptic ulcers and cutaneous inflammations. It is worth mentioning that K. pinnata is present in the National List of Plants of Interest of the Unified Health System - RENISUS (2009). Within this context, the objective of the present study was to characterize the chemical markers in the leaf juices of both species and to evaluate the gastroprotective and topical anti-inflammatory activities. Phytochemical characterization was performed by Thin Layer Chromatography (TLC) and Ultra High Performance Liquid Chromatography coupled to Mass Spectrometer (UHPLC- MS). Gastroprotective activity was evaluated in ethanol and indomethacin induced acute ulcer models, whereas gastric secretion was evaluated in the pylorus ligature model in Wistar rats. Pre-treatment was performed with the juices at the doses of 125, 250 and 500 mg/kg and ranitidine (50 mg/kg) orally. The topical anti-inflammatory activity was evaluated in the carrageenan induced paw edema model and croton oil-induced ear edema in Swiss mice using gel formulations containing the juices at different concentrations (1,25%, 2,5% and 5%) and as the standard drug dexamethasone (1mg/g), all administered topically immediately after induction. The TLC analysis revealed the presence of flavonoid stains in the juices of both species after revelation with the Natural Reagent A, being observed that the two species have different flavonoid profiles. In the analysis by UHPLC-MS the K. brasiliensis leaf juice showed glycosylated flavonoids derived mainly from patuletin, while that of K. pinnata presented glycosylated flavonoids derived mainly from quercetin. The pre-treatment with the K. brasiliensis leaf juice at doses of 125 mg/kg (P<0,01), 250 mg/kg and 500 mg/kg (P<0,001) and K. pinnata at doses of 125 mg/kg (P<0,01), 250 mg/kg and 500 mg/kg (P<0,001) significantly reduced the lesions compared to the positive control in the ethanol induction model. In the indomethacin induction model, the K. brasiliensis leaf juice showed significant results at doses of 250 mg/kg (P<0,05) and 500 mg/kg (P<0,01) and K. pinnata at doses of 250 and 500 mg/kg (P<0,001). Reduction of lesions was accompanied by an increase in total glutathione content and reduction of malondialdehyde levels. In addition, levels of myeloperoxidase, IL-1? and TNF-? were reduced. Cytoprotective effect was also observed in histological evaluation with H&E and maintenance of mucus production with PAS, as well as reduction of iNOS and NF-?B p65 expression and increased expression of ZO-1 by immunohistochemistry. Leaf juices from both species did not change the acidity, pH and volume of the gastric juice. In the ear edema model, the formulations containing the three concentrations of the K. brasiliensis leaf juice significantly reduced the edema when compared to the placebo group (1,25% P<0,05, 2,5% P<0,01 and 5% P<0,01). However, only the formulation containing the juice of the K. pinnata leaf juice at 5% concentration showed a significant result (P<0,01). In the paw edema model, the formulations containing the K. brasiliensis leaf juice at concentrations of 1,25 and 2,5% significantly reduced (P<0,05) the edema in the time 4 h. The formulation at 5% concentration significantly reduced edema at 1 h (P <0,001), 2 h, 3 h and 4 h (P <0,01). Regarding the formulations containing the K. pinnata leaf juice, the concentration of 1,25% significantly reduced the edema in the time 1 h (P<0,01) and 2 h (P<0,05), in the concentration of 5% significantly reduced in time 1 h (P<0,05). The decrease in edema was followed by reduction of miloperoxidase. It was concluded that the juices of both species presented gastroprotective and topical anti-inflammatory activity in vivo models, results that justify the popular use of the species.