Student Number : 0000127R - MPharm dissertation - School of Pharmacy and Pharmacology - Faculty of Health Sciences / Traditional medicines form a significant part of the lives of many people around the world and in South Africa almost 60 % of people consult traditional healers in addition to the modern medical services available. Plants form a significant part of traditional healing and hence, selected species of a traditionally used plant genus, Hermannia, were chosen for biological and chemical investigation to determine a scientific basis for the traditional use of these plants. A phytochemical investigation was carried out, firstly using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) and then isolation and identification of compounds from various Hermannia species. TLC analysis indicated significant similarities between the various species with only H. saccifera displaying chemical anomalies. This was further corroborated by the HPLC analysis although very conservative profiles were produced. Isolation of compounds from H. saccifera yielded a novel labdane compound, E-17, 19-diacetoxy - 15 - hydroxylabda - 7,13 - diene, as well as two flavones, 5,8- dihydroxy-6,7,4’- trimethoxyflavone and cirsimaritin which have previously been isolated. In addition, two commonly found compounds, lupeol and β- sitosterol were isolated from H. cuneifolia and H. salviifolia respectively. This is the first report on the isolation and identification of all five compounds from Hermannia species. Antimicrobial activity was assessed using two methods i.e. minimum inhibitory concentrations as well as the death kinetics assay. Minimum inhibitory concentrations were determined using four Gram-positive and two Gram-negative bacteria as well as two yeasts. All species investigated indicated antimicrobial activity with H. saccifera showing good activity against S. aureus and B. cereus. E-17, 19-diacetoxy - 15 - hydroxylabda - 7,13 - diene isolated from H. saccifera indicated activity (MIC = 23.6 μg/ml against S.aureus) although the activity was less than that of the crude extract (MIC = 19.5 μg/ml), thus, demonstrating that there are a number of compound contributing to the promising activity of the crude extract. This was further corroborated by the bioautograms developed of the H. saccifera extract. Time-kill studies on H. saccifera against S. aureus indicated that at concentrations of 0.1, 0.25 and 0.5 % bacteriostatic activity was observed while at 0.75% the extract achieved complete bactericidal activity after 240min. Free radical scavenging activity was assessed using the 2,2-diphenyl-1-picrylhydrazy (DPPH) and 2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) assays. Ten of the twelve species indicated good activity with H. cuneifolia demonstrating the most promising activity (IC50 = 10.26 μg/ml for DPPH and 10.32 μg/ml for ABTS). Two of the isolated compound, 5,8- dihydroxy-6,7,4’- trimethoxyflavone and cirsimaritin displayed insignificant activity. The 5-lipoxygenase assay was used to assess the anti-inflammatory activity of Hermannia species. All species exhibited intermediate activity with the exception of H. cuneifolia (IC50 = 15.32 μg/ml). In addition, four isolated compounds, 5,8- dihydroxy-6,7,4’- trimethoxyflavone, cirsimaritin, lupeol and β-sitosterol showed moderate inhibition of the enzyme indicating that while these compounds do contribute to the activity of the extracts they are not individually responsible for any significant activity. Antimalarial activity was assessed using the titrated hypoxanthine incorporation assay while toxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay. Only three species indicated any good antimalarial activity i.e. H. saccifera, H. muricata and mostly H. trifurca (IC50 = 25.30, 28.17 and 18.80 μg/ml respectively). However, the activity of H. saccifera and H. trifurca are probably due to a general cytotoxicity as these species exhibited a low safety index. All other species appear safe for use. Several Hermannia species have indicated in vitro biological activity in a number of assays which is related to their use in traditional medicines to treat a number of disease states. Hence, a scientific basis, albeit in vitro, has been established for the use Hermannia species in traditional healing.

Mikania é o maior gênero da tribo Eupatorieae, com cerca de 430 espécies, sendo amplamente distribuído no Brasil, com aproximadamente 200 espécies descritas. Dentro deste gênero, Mikania cordifolia, M. micrantha e M. glomerata são comumente chamadas de guaco e estão entre as espécies mais usadas popularmente, principalmente para o tratamento de febre, reumatismo, doenças do trato respiratório e contra picadas de cobra. No presente trabalho, foram realizados estudos fitoquímicos destas três espécies e foram analisados os efeitos dos tipos de adubação, da radiação solar e do estresse hídrico sobre a produção de metabólitos secundários. Primeiramente, através de estudos fitoquímicos, foram identificados 62 compostos. As frações polares das espécies apresentaram como compostos majoritários derivados fenilpropanóicos, destacando-se o Ácido 3,5-dicafeoilquínico em M. glomerata e M. micrantha e o Ácido Fertárico em M. cordifolia. Na espécie M. glomerata também foram identificados derivados do Ácido glucárico di e tri esterificados por ácidos caféicos, até o momento não descritos na literatura, além de flavonóides sulfatados, os quais também estão presentes em M. micrantha. Nas frações apolares foi verificada a presença de triterpenos, principalmente em M. cordifolia e derivados do Ácido Caurenóico em M. glomerata e M. micrantha. As lactonas sesquiterpênicas foram encontradas em M. cordifolia (esqueleto melampolido) e em M. micrantha (esqueleto micranolido). A análise dos cultivares de M. glomerata e M. cordifolia, revelou uma grande influência das condições de cultivo na composição dos metabólitos secundários. Os experimentos mostraram uma grande variação entre a concentração dos compostos presentes nas duas espécies de Mikania, sendo verificado que as condições de cultivo e principalmente as taxas de luminosidade são de extrema importância na acumulação desses metabólitos. O estresse hídrico não apontou efeitos significativos no acúmulo de substâncias em M. glomerata. As análises histoquímicas se mostraram eficazes na obtenção de informações a respeito da localização de compostos fenólicos nas folhas de M. glomerata. / Mikania is the largest gender of the tribe Eupatorieae, with about 430 species, being distributed thoroughly in Brazil, with approximately 200 described species. In this gender, Mikania cordifolia, M. micrantha and M. glomerata are commonly called \"guaco\" and are among the more popularly used species, mainly for the treatment of fever, rheumatism, diseases of the breathing tract and against snake bites. In the present work, phytochemical studies of these three species were carried out and the effects of the fertilization types, the solar radiation and the hydric stress upon the production of secondary metabolites were analyzed. Firstly, through phytochemical studies, 62 compounds were identified. The polar fractions of the species presented as majority compounds phenylpropanoic derivatives: 3,5-Dicaffeoylquinic acid in M. glomerata and M. micrantha and Fertaric acid in M. cordifolia. In M. glomerata were also identified glucaric acid derivatives di- and tri-esterified with cafeic acids, which are no described in the literature until the moment, besides sulfated flavonoids, which are also present in M. micrantha. In the apolar fractions the triterpenes presence were verified mainly in M. cordifolia and kaurenoic acid derivatives in M. glomerata and M. micrantha. The sesquiterpene lactones were found in M. cordifolia (melampolide skeleton) and in M. micrantha (micranolide skeleton). The analysis of M. glomerata and M. cordifolia cultivars revealed a great influence of the cultivation conditions in the secondary metabolites composition. The experiments showed a great variation among the concentration of the compounds in the two species of Mikania, being verified that the cultivation conditions and mainly the taxes of solar radiation are of extreme importance in the accumulation of these metabolites. The hydric stress didnt have significant effects in the accumulation of substances in M. glomerata. The histochemical analyses were effective in supply information regarding location of phenolic compounds in the leaves of M. glomerata.

Neste estudo foram obtidos os extratos etanólicos das espécies Mikania parodii Cabrera e Mikania pilosa Baker, os quais foram submetidos a fracionamento utilizando as técnicas de cromatografia líquida a vácuo, em coluna clássica e por exclusão. A partir da espécie Mikania parodii foram isolados e identificados através de comparação dos dados dos espectros de RMN 1H e 13C com a literatura os triterpenos acetato de taraxasterol e acetato de pseudotaraxasterol e os flavonóides caempferol-3-O-glicosídeo e quercetina-3-O-glicosídeo e a partir da espécie Mikania pilosa foi isolado e identificado o flavonóide quercetina 7-metil éter. Os extratos e frações de ambas as plantas foram analisados em técnicas hifenadas (CGEM, CLAE-DAD-EM/EM), permitindo a identificação de 46 substâncias em Mikania parodii e 60 substâncias em M. pilosa compreendendo as seguintes classes: monoterpenos, sesquiterpenos, um diterpeno (M. pilosa), triterpenos, flavonóides, derivados do ácido quínico (ácidos clorogênicos), ácidos graxos e seus ésteres. O extrato etanólico bruto de M. parodii apresentou atividade anticonvulsivante e antialérgica. O extrato etanólico bruto de M. pilosa ressuspendido em água : etanol (7:3) inibiu em mais de 50% e 59% respectivamente as células HCT-8 (carcinoma de cólon) e SF925 glioblastoma humano). A análise dos extratos polares de M. camporum e M. vitifolia em CLAE-DAD-EM/EM em comparação com dados da literatura permitiu a identificação dos derivados do ácido quínico: 5-O-Ecafeoilquínico, 3,5-di-O-E-cafeoilquínico, 4,5-di-O-E-cafeoilquínico e de classes de flavonóides apresentando substituintes metila e hexoses em ambas as espécies. / In this study the ethanolic extracts from the species Mikania parodii Cabrera and Mikania pilosa Baker were obtained and fractionated by vacuum liquid chromatography, column chromatography and size-exclusion chromatography. From the species M. parodii were isolated and identified through the comparison of the 1H and 13C NMR spectral data with those in the literature the triterpenes taraxasterol acetate, pseudotaraxasterol acetate and the flavonoids kaempferol-3-O-glucoside and quercetin-3-O-glucoside. From the species Mikania pilosa was isolated the flavonoid quercetin 7-methyl ether. The extracts and fractions of both plants were analyzed by hyphenated techniques (GC-MS and LC-MS/MS), allowing the identification of 46 compounds in M. parodii and 60 compounds in M. pilosa, comprised the classes: monoterpenes, sesquiterpenes, diterpene (M. pilosa), triterpenes, flavonoids, quinic acid derivatives (chlorogenic acids), fatty acids and esters. The ethanolic crude extract of M. pilosa, ressuspended in water:ethanol (7:3) inhibited more than 50 % and 59% respectively the HCT-8 (colon carcinoma) and SF925 cells (human glioblastome). The analysis of polar extracts of the M. camporum and M. vitifolia species in LC-MS/MS in comparison with literature allow the identification of quinic acid derivatives: 5-O-caffeoylquinic acid; 3,5-di-O-caffeoylquinic acid; 4,5-di-O-caffeoylquinic acid, methyl and O-glucosylflavonoids in both species.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-03-18T11:49:47Z No. of bitstreams: 1 PDF - Davy Macgyver da Silva Messiades.pdf: 1230403 bytes, checksum: 455cb9c8a0edaf8b0d94fc14de70dee3 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-22T15:02:15Z (GMT) No. of bitstreams: 1 PDF - Davy Macgyver da Silva Messiades.pdf: 1230403 bytes, checksum: 455cb9c8a0edaf8b0d94fc14de70dee3 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-22T15:02:24Z (GMT) No. of bitstreams: 1 PDF - Davy Macgyver da Silva Messiades.pdf: 1230403 bytes, checksum: 455cb9c8a0edaf8b0d94fc14de70dee3 (MD5) / Made available in DSpace on 2016-07-22T15:02:24Z (GMT). No. of bitstreams: 1 PDF - Davy Macgyver da Silva Messiades.pdf: 1230403 bytes, checksum: 455cb9c8a0edaf8b0d94fc14de70dee3 (MD5) Previous issue date: 2014-06-06 / Aspidosperma pyrifolium Mart. is a species that occurs in the caatinga, being popularly known as Pereiro. This species is economically important to have multiple uses, primarily for use as an ornamental plant (even in recovery of riparian vegetation), as wood (carpentry, charcoal and firewood) and as fodder. Phytochemical studies revealed the presence of indole alkaloids, which were attributed hypotensive, antimicrobial, trypanosomicide and cytotoxic activities. The objective of this research was to realize a phytochemical, toxicological and biological study of A. pyrifolium Mart. The extracts were produced by different extraction techniques and by three different hydroalcoholic concentrations and of proportion of plant material. To perform the first biological activity a factorial design 3 3 was used to order to determine the extract of best antimicrobial action against the strains Escherichia coli (ATCC 25922) , Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Klebsiella pneumoniae (ATCC 4352). This extract was used in the realization of the other biological studies. The results showed that the best antimicrobial activity was obtained with the extract produced by turbólise using 30 g of plant material dissolved in solution of 90% ethanol/water. The phytochemical studies showed the presence of phenolic compounds and saponins. In pharmacological testing was observed that the extract, in the doses tested, exhibited an analgesic and anti-inflammatory potential, and although the crude extract has shown signs of toxicity, these were not evidenced in the pharmacological assays. / Aspidosperma pyrifolium Mart. é uma espécie que ocorre na caatinga, sendo popularmente conhecida como Pereiro. Essa espécie é economicamente importante por ter multiplicidade de usos, principalmente na utilização como planta ornamental (inclusive em processos de recuperação de mata ciliar), como madeira (carpintaria, carvão vegetal e lenha) e como forragem. Estudos fitoquímicos mostraram a presença de alcaloides indólicos, aos quais foram atribuídas atividades hipotensoras, antimicrobianas, tripanossomicidas e citotóxicas. Assim, o objetivo dessa pesquisa foi realizar um estudo fitoquímico, biológico e toxicológico da A. pyrifolium Mart. Os extratos foram produzidos por diferentes técnicas de extração e por três distintas concentrações hidroalcoólicas e de proporção de material vegetal. Para a realização da primeira atividade biológica foi utilizado um planejamento fatorial 3 3 , com a finalidade de se determinar o extrato de melhor ação antimicrobiana frente às cepas Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) e klebsiella pneumoniae (ATCC 4352). Este extrato foi utilizado na realização dos demais estudos biológicos. Os resultados mostraram que a melhor atividade antimicrobiana foi obtida com o extrato produzido por turbólise, utilizando 30 g de material vegetal dissolvido em solução de 90% de etanol/água. O estudo fitoquímico mostrou a presença de compostos fenólicos e saponinas. Nos ensaios farmacológicos observou-se que o extrato, nas doses testadas, apresentou potencial analgésico e anti-inflamatório, e embora o extrato bruto tenha apresentado indícios de toxicidade, estes não foram evidenciados nos ensaios farmacológicos.

The research presented in this thesis addressed knowledge gaps for three medicinal plant taxa, Souroubea spp. (Marcgraviaceae) and Platanus (Platanaceae) as well as Echinacea spp. (Asteraceae). The primary pharmacological mechanism of Souroubea sympetala and Platanus occidentalis were well established, with pentacyclic triterpenes identified as major active principles. My results indicate that major triterpenoids, and crude plant extracts, selectively inhibited monoacyglycerol lipase (MAGL) activity but not fatty acid amide hydrolase (FAAH) activity. These data suggest a possible secondary anxiolytic mechanism of action through the endocannabinoid system (ECS). My study of herb-drug interactions of Souroubea and Plantanus products showed some potential risk when combined with a classic benzodiazepine class drug, diazepam, and I proposed a mechanism through in vitro CYP450 enzyme inhibition. The pharmacokinetic study revealed the difficulty of detecting betulinic acid in animal blood. To support the development a commercial botanical composed of these medicinal plants, an extraction method and a highly sensitive and selectivity HPLC-APCI-MS based quantification method was successfully developed and validated. Part II of this thesis focused on the impact of phytochemical variation and hepatic metabolism on the ECS activity of Echinacea spp. and explored the potential for new applications of Echinacea spp. as a natural health product. My research indicated that considerable variability in the content of phenolic and alkylamide (AKA) compounds reflected similar variability in in vitro bioactivity at ECS-related pharmacological targets. Following biochemometric analysis, several phenolic compounds and AKAs in Echinacea spp. were found to be significant independent variables determining FAAH inhibition and CB receptor activation. Hepatic metabolism was also found to affect the FAAH inhibition of AKA, as increased FAAH inhibitory effects were observed after CYP450-mediated metabolism of both individual AKAs and crude extracts of E angustifolia and E. purpurea, suggesting a “pro-drug” mechanism. Dose dependent activities were observed with oral administration of both E angustifolia and E. purpurea root extract in rat paw model of inflammation and pain. Further tests indicated these activities can be partially blocked by co-administration of CB1 and CB2 receptor antagonists AM251 and AM630, respectively. This evidence suggests activity for peripheral pain was at least partially mediated through the ECS.

The goal of this thesis was to understand and quantify to what extent plants are used for the treatment of mental and folk illnesses such as susto and mal aire in the Neotropics and to investigate the anxiolytic and antiepileptic potential of previously unstudied Neotropical members of the genera Piper and Peperomia. Firstly, the literature was reviewed and a regression analysis method was used in order to quantitatively determine which plant families are preferred for the treatment of mental, behavioral and neurological health disorders in the Neotropics. This analysis identified Piperaceae, among others, as an important taxonomic group for the treatment of such disorders. Following that lead, a botanical survey was conducted in Peru, where 47 species of Piperaceae and 21 plants traditionally used for folk illnesses by the Yanesha of Peru, an Amazonian ethnic group, were collected. In order to target potential anxiolytic and antiepileptic plants, two high throughput bioassays were used to evaluate the extracts’ in vitro activity on the γ-aminobutyric acid (GABA) system. Plant extracts in general demonstrated moderate to high affinity to the GABA-BZD receptor. Additionally, extracts demonstrated low to moderate activity in the inhibition of the GABA-T enzyme, with a few plants exhibiting promising activity. Plants selected by the Yanesha showed comparable activity to the other Piperaceae plants with Piper cremii being the most active plant in the GABAA assay, and Drymaria cordata in the GABA-T assay. Finally, four phytochemicals from Piper tuerckheimii, a plant regarded as one of the most effective traditional remedy for the treatment of epilepsy and susto by the Q’eqchi’ Maya of Belize presented, were isolated for the first time.

The goal of this thesis was to understand and quantify to what extent plants are used for the treatment of mental and folk illnesses such as susto and mal aire in the Neotropics and to investigate the anxiolytic and antiepileptic potential of previously unstudied Neotropical members of the genera Piper and Peperomia. Firstly, the literature was reviewed and a regression analysis method was used in order to quantitatively determine which plant families are preferred for the treatment of mental, behavioral and neurological health disorders in the Neotropics. This analysis identified Piperaceae, among others, as an important taxonomic group for the treatment of such disorders. Following that lead, a botanical survey was conducted in Peru, where 47 species of Piperaceae and 21 plants traditionally used for folk illnesses by the Yanesha of Peru, an Amazonian ethnic group, were collected. In order to target potential anxiolytic and antiepileptic plants, two high throughput bioassays were used to evaluate the extracts’ in vitro activity on the γ-aminobutyric acid (GABA) system. Plant extracts in general demonstrated moderate to high affinity to the GABA-BZD receptor. Additionally, extracts demonstrated low to moderate activity in the inhibition of the GABA-T enzyme, with a few plants exhibiting promising activity. Plants selected by the Yanesha showed comparable activity to the other Piperaceae plants with Piper cremii being the most active plant in the GABAA assay, and Drymaria cordata in the GABA-T assay. Finally, four phytochemicals from Piper tuerckheimii, a plant regarded as one of the most effective traditional remedy for the treatment of epilepsy and susto by the Q’eqchi’ Maya of Belize presented, were isolated for the first time.

For over a century, documented scientific research and debate has revolved around the therapeutic properties of the medicinal plant Echinacea angustifolia. With overwhelming evidence demonstrating the biological activity of its root phytochemical constituents, the genetic improvement of E. angustifolia by selecting phytochemically rich genotypes has garnered both scientific and commercial interest. This dissertation presents results of multi-disciplinary experiments intended to help establish scientifically based guidelines for breeding efforts aimed at developing therapeutically superior varieties of E. angustifolia in British Columbia. Cultivated E. angustifolia populations from British Columbia and Washington were grown in a common greenhouse environment to identify possible genetically superior populations with respect to root concentrations of therapeutically relevant caffeic acid derivatives (CAD) and alkamides. However, none of the studied cultivated E. angustifolia populations showed significant genetic differences in terms of root phytochemical traits. In the second part, an investigation into correlations between root and shoot phytochemical concentrations in field- and greenhouse-grown plants revealed that concentrations of therapeutically relevant marker compounds in shoots were generally poor predictors of concentrations in roots. Some weak yet significant positive correlations were observed between root and shoot concentrations of CADs but were inconsistent between the two environments. Significant genotype by environment interactions were documented for the first time in phytochemical traits of E. angustifolia in a study of five genetically homogeneous populations grown in three different environments, including 1 greenhouse and 2 field sites in British Columbia. For the final objective, in vitro bioassays showed that environmentally and genotypically related differences in concentrations of CADs and alkamides in E. angustifolia ethanolic root extracts did not translate into significant differences in their anti-inflammatory potential as measured by pro-inflammatory interleukin (IL-6 and IL-8) secretion in human bronchial epithelial (BEAS-2B) cells challenged with rhinovirus. When used in isolation however, pure tetraene alkamide showed a significant inhibitory effect on secretion, thereby further supporting the use of high alkamide production as a selection criterion for therapeutic E. angustifolia cultivar development. A series of recommendations derived from these findings are presented along with ideas for important future studies in the field of Echinacea research.

The goal of this thesis was to understand and quantify to what extent plants are used for the treatment of mental and folk illnesses such as susto and mal aire in the Neotropics and to investigate the anxiolytic and antiepileptic potential of previously unstudied Neotropical members of the genera Piper and Peperomia. Firstly, the literature was reviewed and a regression analysis method was used in order to quantitatively determine which plant families are preferred for the treatment of mental, behavioral and neurological health disorders in the Neotropics. This analysis identified Piperaceae, among others, as an important taxonomic group for the treatment of such disorders. Following that lead, a botanical survey was conducted in Peru, where 47 species of Piperaceae and 21 plants traditionally used for folk illnesses by the Yanesha of Peru, an Amazonian ethnic group, were collected. In order to target potential anxiolytic and antiepileptic plants, two high throughput bioassays were used to evaluate the extracts’ in vitro activity on the γ-aminobutyric acid (GABA) system. Plant extracts in general demonstrated moderate to high affinity to the GABA-BZD receptor. Additionally, extracts demonstrated low to moderate activity in the inhibition of the GABA-T enzyme, with a few plants exhibiting promising activity. Plants selected by the Yanesha showed comparable activity to the other Piperaceae plants with Piper cremii being the most active plant in the GABAA assay, and Drymaria cordata in the GABA-T assay. Finally, four phytochemicals from Piper tuerckheimii, a plant regarded as one of the most effective traditional remedy for the treatment of epilepsy and susto by the Q’eqchi’ Maya of Belize presented, were isolated for the first time.

<p>Since Helichrysum is known by the indigenous people of Africa for therapeutic properties, such as against colds, flu and wounds, the aim of this study was to focus on the patulum species found predominantly in the Western Cape region of South Africa and by means of isolation and identification of the plant constituents, be able to relate the therapeutic activity on the basis of literature precedents, to the compounds extracted.</p>