Conselho Nacional de Desenvolvimento Científico e Tecnológico / Spilanthes Acmella (L.) Murray (Asteraceae) is native to South America, popularly known as jambu. This plant is used in Northern Region of Brazil as a condiment in typical dishes such as tacacá and pato no tucupi and has application in traditional medicine to treat toothache, stomatitis and throat diseases. Furthermore, several therapeutic properties have been reported for S. acmella such as antiviral, antibacterial, antiseptic, diuretic, anti-inflammatory and healing agent. Spilanthol is the major constituent from flowers of this species, which is associated with several biological activities: analgesic, larvicide, insecticide, antibacterial, antifungal, etc. Currently, extracts of jambu and spilanthol are also used as ingredients for cosmetic anti-aging. Given the above, this work reports the development of a HPLC-DAD method for determination of spilanthol in samples of S. acmella. This study used samples from accessions of S. acmella (fresh whole plant and its parts: leaves, stems and flowers, fresh and dried) cultivated and collected in the Rural Campus-UFS. The conditions for analysis using HPLC-DAD system Shimadzu LC 20A Prominence were: analytical column Luna ® C18 (250 mm x 4.6 mm, 5 m) Phenomenex, gradient elution using as mobile phase MeOH (B): H2O (A): 85% (A) for 6 min., 85-100% (A) for 1 min., 100% (A) for 5 min.; 100-85% (A) for 1 min.; 85% (A) for 10 min; flow rate 1.0 mL/min, injection volume 20 L and the chromatograms obtained at =230 nm. Spilanthol was used as a reference standard (tr=5.1 min). The method was validated according to RE 899/03 (ANVISA). Linearity was determined by external standard calibration curve (r2=0.9997) and by the standard addition method (r2=0.9995). The limit of quantification and detection were 0.7 and 0.1 g/mL, respectively. The repeatability showed coefficients of variation (CV, %) between 0.21 to 2.85% and the accuracy, expressed as a recovery percentage ranged from 100.2 to 103.3%. The method was applied in 41 samples of S. acmella including samples from genetic improvement. Samples of dried flowers and accession J-05 had the highest content of spilanthol (101.2 g/mL and 120.5 g/mL, respectively). Therefore, this work presents a contribution to the quality control of extracts of S. acmella. / Spilanthes acmella (L.) Murray (Asteraceae) é uma planta nativa da América do Sul conhecida popularmente como jambú. Esta planta é usada como condimento em pratos típicos da Região Norte como o tacacá e o pato no tucupi e tem aplicação na medicina tradicional para tratar dor de dente, estomatite e doenças da garganta. Além disso, várias propriedades terapêuticas foram relatadas para S. acmella tais como antiviral, antibacteriana, anti-séptica, diurética, antiinflamatória e cicatrizante. Espilantol é o constituinte majoritário das flores desta espécie, o qual está associado a várias atividades biológicas: analgésica, larvicida, inseticida, antimicrobiana, fungicida, etc. Atualmente, extratos de jambú e o espilantol são usados como ingredientes para cosmético antienvelhecimento. Diante do exposto, este trabalho relata o desenvolvimento de um método analítico por CLAE-DAD para determinação de espilantol em amostras de S. acmella. Neste estudo foram utilizadas amostras dos acessos de S. acmella (planta inteira fresca e suas partes: folhas, caules e inflorescências frescas e secas) cultivadas e coletadas no Campus Rural da UFS. As condições de análise utilizando o sistema CLAE-DAD Shimadzu LC 20A Prominence foram: coluna analítica C18 Luna® (250 mm x 4,6mm, 5 m) Phenomenex; eluição gradiente no modo reverso, utilizando como fase móvel MeOH(B): H2O(A): 85% (A) por 6 min., 85-100% (A) por 1 min., 100% (A) por 5 min.; 100-85% (A) por 1 min.; 85% (A) por 10 min; vazão 1,0 mL/min; volume de injeção 25 μL e os cromatogramas obtidos em =230 nm. Espilantol foi usado como padrão de referência (tr=5,1 min). O método foi validado conforme RE no 899/03 (ANVISA). A linearidade foi determinada pela curva de padrão externo (r2=0,9997) e por adição de padrão (r2=0,9995). O limite de quantificação e de detecção foram 0,7 e 0,1 μg/mL, respectivamente. A repetibilidade apresentou coeficientes de variação (CV%) entre 0,21 a 2,85% e a exatidão, expressa como percentual de recuperação, variou de 100,2 a 103,3%. O método foi aplicado em 41 amostras de S. acmella incluindo amostras oriundas de melhoramento genético. As amostras da flores seca e do acesso J-05 apresentaram o maior teor de espilantol (101,2 μg/mL e 120,5 g/mL, respectivamente). Esse trabalho apresenta uma contribuição ao controle de qualidade de extratos de S. acmella.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2014-01-22T18:12:37Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InvestigacaoEfeitoIctiotoxico.pdf: 11184223 bytes, checksum: 68fd5d52e27da07ce25ea581d47609d0 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-01-24T15:12:32Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InvestigacaoEfeitoIctiotoxico.pdf: 11184223 bytes, checksum: 68fd5d52e27da07ce25ea581d47609d0 (MD5) / Made available in DSpace on 2014-01-24T15:12:32Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InvestigacaoEfeitoIctiotoxico.pdf: 11184223 bytes, checksum: 68fd5d52e27da07ce25ea581d47609d0 (MD5) Previous issue date: 2013 / Dentre as várias espécies de plantas medicinais, encontra-se a espécie Spilanthes acmella, conhecida popularmente como jambú que se destaca por apresentar inúmeras aplicações na área da medicina popular. A medicina tradicional recomenda suas folhas e flores na elaboração de infusões no tratamento de anemia, dispepsia, malária, afecções da boca (dor de dente) e da garganta, contra escorbuto e também como antibiótico e anestésico. Sendo seus principais efeitos atribuídos ao espilantol, que é um representante importante das substâncias presentes nessas plantas. Alguns estudos já foram realizados utilizando o espilantol, possibilitando algumas informações da ação dessa substância, como seu efeito e imunomodulador devido sua interação funcional com monócitos, granulócitos e células killers. Porém, ainda não existem estudos eletrofisiológicos acerca de sua ação ictiotóxica, utilizando, por exemplo, o eletroencefalograma para demonstrar sua ação ao nível de Sistema Nervoso Central ou eletromiograma para verificar a ocorrência de sua ação a nível muscular no Zebrafish, evocando a necessidade dessa pesquisa a respeito do assunto. Com base nisso, o presente trabalho objetivou investigar a ação ictiotóxica do extrato etanólico da raiz de Spilanthes acmella em Zebrafish através da análise eletrofisiológica e comportamental. Os resultados mostraram que o extrato etanólico de Spilanthes acmella é um potente indutor de excitabilidade central no zebrafish, sendo isso constatado a partir das mudanças de padrões de atividade elétrica vistas no eletroencefalograma do animal submetido à droga e através do aumento da atividade encefálica visto no espectograma. O extrato também causou alterações, em menor escala, nos traçados eletromiográficos do zebrafish submetido à mesma concentração da droga, com aparecimento de contrações musculares esparsas e de mioclonias breves. Eos achados comportamentais, a partir da delimitação de três estágios de comportamentos, os quais se iniciaram com o aumento da excitabilidade do animal e culminam com a convulsão e morte do peixe, serviram para corroborar com os achados eletrofisiológicos de que o extrato etanólico de Spilanthes acmellaatua como potente droga com ação no sistema nervoso do zebrafish, com atividade convulsivante. / Among the various species of medicinal plants, we can find the Spilanthes acmella species, popularly known as Jambu that stands out due to its numerous applications in the folk medicine field. Traditional medicine recommends the use of its leaves and flowers in the preparation of infusions to treat anemia, dyspepsia, malaria, mouth diseases (tooth pain) and throat diseases, against scurvy and also as antibiotic and anesthetic, being its main effects attributed to espilantol, which is an important representative of the substances present in these plants. Some studies have been performed using the espilantol, providing some information of the effect of this substance, as its immunomodulatory effect and because of its functional interaction with monocytes, granulocytes and killer cells. However, there are still no electrophysiological studies about its ictiotoxic action using, for example, the EEG to demonstrate its action at the central nervous system level or electromyogram to verify the occurrence of their effects in the Zebrafish muscles, evoking the need for this research. Based on this, the present study aimed to investigate the ictiotoxic action of the ethanol extract of the Spilanthes acmella root in Zebrafish by electrophysiological and behavioral analysis. The results showed that the ethanol extract of Spilanthes acmella is a potent inducer of central excitability in zebrafish, this being evidenced by changes in electrical activity patterns seen in the EEG of the animals subjected to the extract and by increasing brain activity seen in the spectrogram. The extract also caused changes, in a lesser extent, on the electromyographic tracings of zebrafish subjected to the same concentration of the extract, with the appearance of scattered muscle contractions and brief myoclonus. And the behavioral findings from the delimitation of three stages of behavior, which began with the increased excitability of the animal and resulted in the seizure and death of the fish, served to corroborate the electrophysiological findings that the ethanol extract of Spilanthesacmella acts as a potent substance acting on the nervous system of zebrafish, with convulsant activity.

The aim of this study was to isolate and characterize antifungal compounds from the most active medicinal plant species that could be used to address secondary infection problems in immunocompromised patients. An ethnobotanical study was conducted and 45 medicinal plant species used traditionally to treat candidiasis and related infections in HIV/AIDS patients were identified and documented. The most popular plant species used included Acacia caffra, Clerodendrum glabrum, Croton gratissimus, Elaeodendron transvaalense, Faurea saligna, Hippocratea longipetiolata, Osyris lanceolata, Richardia brasiliensis, Schkuhria pinnata, Schotia brachypetala, Spilanthes acmella, Strychnos potatorum, Vangueria infausta subsp. infausta and Withania somnifera. The plant parts used in the therapeutic preparations were roots (26.7%), bark (22.2%), and a combination of roots and bark (17.7%). Decoctions (44.4%), infusions (20%) and macerations (17.7%) were used. Most of the herbal remedies were administered orally. Chemical profiles of the plant species were established by using thin layer chromatography. Leaf extracts of these plant species were tested for antimicrobial activity against two common pathogenic fungal species in humans (Candida albicans and Cryptococcus neoformans) and four nosocomial bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) using a two-fold serial microdilution method and bioautography. All plant species investigated had some degree of antimicrobial activity against the test microorganisms. The hexane and the acetone extracts of Clerodendrum glabrum, Hippocratea longipetiolata, Schkuhria pinnata and Withania somnifera were the most active with MIC values ranging from 0.06 to 0.08 mg/ml. The most susceptible pathogen to the test samples was C. neoformans while C. albicans was resistant to most of the plant extracts. The water extracts of Withania somnifera and Hippocratea longipetiolata (14%) had MIC < 1 mg/ml against C. albicans. C. neoformans was susceptible to nine water plant extracts (64%) with MIC < 1 mg/ml and the promising activity was observed in Hippocratea longipetiolata and Faurea saligna extracts with MIC values of 0.16 and 0.31 mg/ml respectively. The hexane extract of C. glabrum was the most active against C. albicans with an MIC value of 0.06 mg/ml and total activity of 550 ml/g. In the bioautography, most plant extracts tested had few active compounds, others had no active components at all and this may be attributed to the disruption of synergism by the thin layer chromatography. C. glabrum had eight active antifungal compounds on bioautograms and most of these components were observed in the EMW solvent system. Based on this and its wide distribution in rural areas, C. glabrum was chosen for further study. The antioxidant activity and possible immune boosting potential of the species were determined using 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH), 2, 2’ azinobis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays. In the DPPH qualitative assay, the aqueous plant extracts had several prominent antioxidant components than the organic plant extracts. The aqueous plant extracts which had the most prominent antioxidant activity were F. saligna with 8 compounds, followed by E. transvaalense, H. longipetiolata O. lanceolata, R. brasiliensis and S.brachypetala, with five compounds each and their Rf values ranged from 0;06 to 0.94. This appears to validate the ethnomedicinal use of the plant species to some extent because decoction is the most common method used in the preparation of the remedy by the traditional healers. With regard to the organic plant extracts, only one plant extract, F. saligna had two prominent antioxidant components at Rf values 0.81 and 0.88. A third of the plant species had a high level of free radical scavenging activities in the DPPH, ABTS and FRAP assays. However, all plant extracts had lower antioxidant activity than the positive control (Trolox) used. The selected plant species were also evaluated for their in vitro toxicity against the Vero monkey kidney cell line using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The acetone plant extracts of O. lanceolata, S. acmella, S. pinnata and S. brachypetala had high cytotoxic activity against Vero cells with IC50 values of 13.7±0, 19.9±0.001, 21.6±0.001 and 28.34±0.001 μg/ml respectively. However, their IC50 values were higher than that of the positive control, doxorubicin (IC50 = 9.9±0 μg/ml). The rest of the acetone plant extracts (64%) had moderate cytotoxic activity (30 < IC50<100 μg/ml). The aqueous plant extracts were relatively non-toxic to the Vero cells with IC50 values ranging from 137 to > 500 µg/ml. This supports the use of aqueous extracts in the traditional medicine. However, their low selectivity index values ranging from 0.26 to 1.68 suggest that the plant extracts are probably suitable for external use only. Fractionation of the hexane extract of the leaves of C. glabrum by chromatographic techniques yielded six fractions of which fractions C and D had significant antifungal activity (average MIC value = 0.1 mg/ml) against C. albicans and C. neoformans. From these fractions, one new triterpenoid, 3-(1-oxobutyl)-11α-hydroxytaraxast-20(30)-ene-24,28-dioic acid (clerodendrumic acid) (1) was isolated along with known heptadecanoic acid (2). C. albicans was relatively insensitive to clerodendrumic acid (1) (MIC value = 125 µg/mL) and was resistant to heptadecanoic acid (2) (MIC value = 188 µg/ml). Compounds 1 and 2 were non-toxic against monkey kidney Vero cells in vitro with IC50 values of 202.6 and 108.4 µg/ml respectively. Due to its low antifungal activity, the novel compound clerodendrumic acid (1) is not a viable candidate for drug development which could be used to combat candidiasis and related fungal infections. However, due to its relative safety, it may possibly be used as a lead compound to produce new chemically modified active derivatives or could be used together with known antibiotics to mitigate their undesirable side effects. To the best of our knowledge, the isolation of a novel, clerodendrumic acid (1) and a known heptadecanoic acid (2) compounds from leaf extracts of C. glabrum is reported herein for the first time. The results obtained from this study generally substantiate the rationale behind the use of the selected plant species in the traditional medicine to treat candidiasis and related infections to some extent. This study showed the potential of studying traditional medicine in the search for effective plant extracts or new lead compounds that could be developed into drugs for combating microbial infections among the rural poor people. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Paraclinical Sciences / Unrestricted