Objetivo: revisar sistematicamente os efeitos da Spirulina platensis em marcadores inflamatórios em humanos. Métodos: foi realizada busca por ensaios clínicos randomizados, realizados em humanos adultos com o objetivo de verificar os efeitos da Spirulina Platensis sob marcadores inflamatórios. A busca foi efetuada até outubro de 2015 nas bases de dados Medline via Pubmed, Cochrane Central, Clinical Trials, Scielo, LILACS, banco de teses da CAPES, assim como em bancos de grey literature. Resultados: Identificados vinte e oito estudos dos quais foram elegíveis três ensaios clínicos randomizados. A amostra foi constituída de 34 a 78 participantes, de ambos os sexos, com idade variando de 18 a 87 anos. Os marcadores inflamatórios avaliados foram: IL-2, IL-6, e TNF- e a intervenção foi feita com cápsulas de Spirulina platensis versus placebo. A IL-2 foi avaliada em dois estudos porém, não foi possível realizar a metanálise, pois um dos artigos não descrevia os valores encontrados em seu grupo controle. A IL-6 ao ser analisada em conjunto, não demonstrou diferença significativa com o uso da Spirulina platensis [-0,36 (IC95%: -0,90 a 0,18), com I2=0%]. Quanto ao TNF- não houve diferença significativa com o uso da Spirulina platensis [-0,03 (IC95%: -0,42 a 0,37)] com teste de inconsistência mostrando moderada heterogeneidade e insignificância estatística [I2= 31%, P= 0,23]. Conclusão: A informação disponível sobre o tema proposto é escassa e o efeito da Spirulina platensis sobre os marcadores inflamatórios se mostrou inconsistente. Ensaios clínicos randomizados adicionais com maior número de participantes e melhores avaliações dos marcadores são necessários para verificar o real papel anti-inflamatório da Spirulina platensis. / Objective: Perform a systematic review on the anti-inflammatory effects of Spirulina platensis in humans. Methods: A search was conducted in Medline via PubMed, Cochrane Central and Clinical Trials until October 2015 and dissertations published in CAPES, and gray literature banks for randomized controlled trials performed in adults to assess the anti-inflammatory effects of Spirulina platensis. Results: The search identified twenty eight articles. Only three randomized clinical trials were elegible for this study. The sample ranged between 34 and 78 participants, both genders, with the age ranged from 18 to 78 years. The measurements of inflammatory outcome were assessed through IL-2, IL-6, and TNF-. The intervention was capsules of Spirulina platensis versus placebo. The IL-2 was evaluated in two studies but it was not possible to evaluate the results together through meta-analysis because one of the articles did not describe the values in the control group. IL-6, when analyzed together, showed no significant difference between Spirulina platensis and placebo [-0.36 (95% CI: -0.90 to 0.18), with I2 = 0%]. Regarding the TNF- no significant difference was found [-0.03 (95% CI: -0.42 to 0.37)]. The inconsistency test showed moderate heterogeneity and no statistical significance [I2 = 31%, P = 0.23]. Conclusion: The available information on anti-inflammatory effects of S. platensis is scarce. Spirulina platensis presented no significant effect on the inflammatory markers. Additional randomized clinical trials, with more participants and better markers assessments are needed to verify the real anti-inflammatory role of Spirulina platensis.

nÃo hà / A pentoxifilina (PTX) à um inibidor nÃo-seletivo da fosfodiesterase, com aÃÃes antiinflamatÃrias vasculares e reolÃgicas que podem neutralizar algumas das mudanÃas no diabetes mellitus (DM) que contribuem para amenizar os seus efeitos secundÃrios como a neuropatia, a retinopatia e a nefropatia. Tendo em vista as propriedades antiinflamatÃrias da pentoxifilina e o envolvimento da inflamaÃÃo com o DM, buscou-se investigar seus possÃveis efeitos hipoglicemiante e hipolipemiante no modelo de DM induzido por aloxano em ratos. A pentoxifilina (PTX) em estudos pilotos apresentou efeito hipoglicemiante e reduziu os nÃveis de triglicerÃdeos em animais com diabetes induzidos por aloxano, nas doses 5, 25, 50 e 100 mg/Kg. A administraÃÃo oral da associaÃÃo de pentoxifilina (PTX) com glibenclamida (GLI), PTX5 + GLI2, causou reduÃÃes significativas nos nÃveis plasmÃticos de glicose e triglicerÃdeos em curto e longo prazo, evidenciando que o mecanismo de aÃÃo da PTX pode ser explicado via canais de K+ATP-dependentes. A administraÃÃo oral da associaÃÃo de Pentoxifilina (PTX) com Metformina (MET), PTX5 + MET5, ocasionou uma reduÃÃo da hiperglicemia apenas a longo prazo, sugerindo nÃo compartilharem o mesmo mecanismo. A PTX nÃo bloqueou a hiperglicemia induzida pelo Diazoxido (DZD), um antagonista da GLI, que inibe a secreÃÃo de insulina prolongando o tempo de abertura dos canais de K+ATP-dependentes, sugerindo que outros fatores, alÃm do bloqueio de canais de K+-ATP dependentes, podem estar envolvidos. A reduÃÃo nos valores de hemoglobina glicada (A1C) e de frutosamina mostraram que o tratamento com PTX50 e com a associaÃÃo PTX5+GLI2 melhorou o controle glicÃmico dos animais em estudo, indicando que esta droga pode inibir o desenvolvimento de lesÃes micro e macrovasculares advindas do DM. A PTX mostrou um marcante efeito antiinflamatÃrio, melhorando o estado geral dos ratos em experimentaÃÃo. Reduziu, de forma significativa, o edema de pata nas doses de 50 e 100 mg/Kg, todavia foi visto que o perfil inflamatÃrio no rato diabÃtico tem um padrÃo diferenciado do rato normal, evidenciando uma amplificaÃÃo do processo inflamatÃrio no rato diabÃtico quando comparado ao rato normal. Foi visto ainda que os nÃveis de TNF-∝ e IL-6 aumentaram de modo significante apÃs a induÃÃo do edema de pata nos ratos diabÃticos, entretanto nos ratos tratados com PTX os nÃveis teciduais destas citocinas mostraram-se significativamente mais baixos, o que fala a favor de uma evidente aÃÃo antiinflamatÃria da PTX. A PTX mostrou tambÃm um importante efeito antioxidante reduzindo, de forma significativa, as liberaÃÃes de nitrito tecidual e sÃrica, atuando favoravelmente na reduÃÃo de radicais livres. O tratamento prolongado com PTX foi eficaz em manter o padrÃo normal do pÃncreas, do fÃgado e dos rins nos grupos diabÃticos tratados com PTX50 e com PTX5+GLI2, indicando uma aÃÃo protetora da PTX contra a citotoxicidade induzida pelo aloxano. Os efeitos hipoglicemiante e hipotrigliceridÃmico da PTX, aqui demonstrados, podem està correlacionado com sua aÃÃo sobre o estresse oxidativo e sobre a low grade inflammation, o que torna a PTX um importante alvo terapÃutico para o manejo do diabetes mellitus na clÃnica / Pentoxifylline (PTX) is a non-selective inhibitor of phosphodiesterase with anti-inflammatory vascular and rheological properties. The drug can neutralize some of the changes seen in diabetes mellitus (DM), contributing to attenuate diabetes secondary complications as neuropathy, retinopathy and nephropathy. Considering PTX anti-inflammatory properties and the known involvement of inflammation with DM, we investigated its possible hypoglycemic and hypolipidemic effects in the model of alloxan-induced DM in rats. Pentoxifylline pilot studies in reduced plasma levels of glucose and triglycerides in animals with diabetes induced by alloxan at the doses of 5, 25, 50 and 100 mg/kg. Oral administration of the combination of PTX with glibenclamide (GLI), PTX5 + GLI2, caused significant reductions in plasma levels of glucose and triglycerides in the short and long term, indicating that the mechanism of action of PTX can be explained via K+ATP-dependent channels. The oral administration of the combination of pentoxifylline (PTX) with metformin (MET), PTX5+MET5, caused a reduction of only the long term hyperglycemia, suggesting that these two drugs do not share the same mechanism. PTX did not block the hyperglycemia induced by diazoxide (DZD), an antagonist of GLI, which inhibits insulin secretion by prolonging the opening time of the K+ATP-dependent-channel. This result suggests that other factors, in addition to the blockade of the K+ATP dependent channels, may be involved. The reduction in glycosylated hemoglobin (A1C), and fructosamine showed that treatment with the combination PTX5+GLI2 and PTX50, improved glycemia in the study, indicating that this drug can inhibit the development of macrovascular and microvascular injury resulting from DM. The PTX showed a marked anti-inflammatory effect, improving the general condition of rats subjected to acute inflammation models. PTX reduced significantly, the paw edema at doses of 50 and 100 mg / kg, However, the inflammatory profile in diabetic rats have a different pattern of that seen in non-diabetic rat, showing an amplification of the inflammatory process. We showed that the levels of TNF-α and IL-6 were significantly increased after the induction of paw edema in diabetic rats, but in the rats treated with PTX tissue levels of these cytokines were significantly lower, which indicating a clear anti-inflammatory action of PTX. PTX also showed a significant antioxidant effect reducing significantly the release of tissue and serum nitrite, acting favorably in the reduction of free radicals. The prolonged treatment with PTX was effective in maintaining the normal histological pattern of the pancreas, liver and kidneys in diabetic groups treated with PTX50 PTX5 + and GLI2, indicating a protective effect of PTX against alloxan-induced cytotoxicity. The hypoglycemic and hypotriglyceridemic effects of PTX, shown here, may correlate with its effect on oxidative stress and on low grade inflammation, making PTX an important candidate for the management of diabetes mellitus in the clinic

Objetivo: revisar sistematicamente os efeitos da Spirulina platensis em marcadores inflamatórios em humanos. Métodos: foi realizada busca por ensaios clínicos randomizados, realizados em humanos adultos com o objetivo de verificar os efeitos da Spirulina Platensis sob marcadores inflamatórios. A busca foi efetuada até outubro de 2015 nas bases de dados Medline via Pubmed, Cochrane Central, Clinical Trials, Scielo, LILACS, banco de teses da CAPES, assim como em bancos de grey literature. Resultados: Identificados vinte e oito estudos dos quais foram elegíveis três ensaios clínicos randomizados. A amostra foi constituída de 34 a 78 participantes, de ambos os sexos, com idade variando de 18 a 87 anos. Os marcadores inflamatórios avaliados foram: IL-2, IL-6, e TNF- e a intervenção foi feita com cápsulas de Spirulina platensis versus placebo. A IL-2 foi avaliada em dois estudos porém, não foi possível realizar a metanálise, pois um dos artigos não descrevia os valores encontrados em seu grupo controle. A IL-6 ao ser analisada em conjunto, não demonstrou diferença significativa com o uso da Spirulina platensis [-0,36 (IC95%: -0,90 a 0,18), com I2=0%]. Quanto ao TNF- não houve diferença significativa com o uso da Spirulina platensis [-0,03 (IC95%: -0,42 a 0,37)] com teste de inconsistência mostrando moderada heterogeneidade e insignificância estatística [I2= 31%, P= 0,23]. Conclusão: A informação disponível sobre o tema proposto é escassa e o efeito da Spirulina platensis sobre os marcadores inflamatórios se mostrou inconsistente. Ensaios clínicos randomizados adicionais com maior número de participantes e melhores avaliações dos marcadores são necessários para verificar o real papel anti-inflamatório da Spirulina platensis. / Objective: Perform a systematic review on the anti-inflammatory effects of Spirulina platensis in humans. Methods: A search was conducted in Medline via PubMed, Cochrane Central and Clinical Trials until October 2015 and dissertations published in CAPES, and gray literature banks for randomized controlled trials performed in adults to assess the anti-inflammatory effects of Spirulina platensis. Results: The search identified twenty eight articles. Only three randomized clinical trials were elegible for this study. The sample ranged between 34 and 78 participants, both genders, with the age ranged from 18 to 78 years. The measurements of inflammatory outcome were assessed through IL-2, IL-6, and TNF-. The intervention was capsules of Spirulina platensis versus placebo. The IL-2 was evaluated in two studies but it was not possible to evaluate the results together through meta-analysis because one of the articles did not describe the values in the control group. IL-6, when analyzed together, showed no significant difference between Spirulina platensis and placebo [-0.36 (95% CI: -0.90 to 0.18), with I2 = 0%]. Regarding the TNF- no significant difference was found [-0.03 (95% CI: -0.42 to 0.37)]. The inconsistency test showed moderate heterogeneity and no statistical significance [I2 = 31%, P = 0.23]. Conclusion: The available information on anti-inflammatory effects of S. platensis is scarce. Spirulina platensis presented no significant effect on the inflammatory markers. Additional randomized clinical trials, with more participants and better markers assessments are needed to verify the real anti-inflammatory role of Spirulina platensis.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O Ãleo-resina da Copaifera langsdorffii (Leguminaceae) Ã utilizado popularmente no tratamento de processos inflamatÃrios e na cicatrizaÃÃo de feridas e Ãlceras. Estudos prÃvios estabeleceram as propriedades gastroprotetora e antiinflamatÃria em modelos animais. O presente estudo avaliou o potencial antiinflamatÃrio do Ãleo-resina da Copaifera langsdorffii (ORCL) e de seu constituinte diterpÃnico, Ãcido kaurenÃico (AK) nos modelos experimentais de colite induzida por Ãcido acÃtico (UC-AA), por Ãcido trinitrobenzÃnico sulfÃnico (UC-TNBS), e ainda, indometacina e isquemia-reperfusÃo induzindo inflamaÃÃo intestinal (II-IND e II-I/R). E tambÃm, o potencial de cicatrizaÃÃo de feridas foi avaliado em ratos com feridas abertas e com incisÃo. Ratos foram prÃ-tratados via oral (15 e 2 horas antes) ou retal 2 horas apÃs a induÃÃo da colite, com ORCL (200 e 400mg/Kg), AK (50 e 100mg/Kg) ou veÃculo (1mL, 2% Tween 80 ou 1mL, 2% DMSO). A colite foi induzida pela aplicaÃÃo de 2mL de Ãcido acÃtico 4% (v/v) ou TNBS (0,25 mL com 20mg) e 24 ou 72 horas depois, os danos na mucosa do cÃlon foram avaliados, medidos os nÃveis de mieloperoxidase e malonaldeÃdo. No modelo de (CU-AA), houve uma importante reduÃÃo no escore da lesÃo e peso Ãmido nos animais tratados com as substÃncias teste, quando comparado ao controle veÃculo. Os efeitos foram confirmados na bioquÃmica pela significante reduÃÃo da atividade da mieloperoxidase (MDA), o marcador da infiltraÃÃo de neutrÃfilos e pela marcada diminuiÃÃo nos nÃveis de malonaldeÃdo, um indicador da lipoperoxidaÃÃo. AlÃm de o Ãcido acÃtico aumentar os nÃveis de nitrito e da enzima catalase no cÃlon, onde o tratamento com ORCL diminuiu significativamente. A anÃlise microscÃpica revelou uma diminuiÃÃo da infiltraÃÃo de cÃlulas inflamatÃrias e do edema da submucosa, nos segmentos do cÃlon tratados com ORCL ou AK. De maneira similar, no modelo de UC-TNBS, houve uma reduÃÃo do escore da lesÃo e peso Ãmido do cÃlon de animais prÃ-tratados com ORCL (400mg/Kg, v.o. ou retal) 2, 24 e 48 horas apÃs a injeÃÃo intracolÃnica de TNBS. A atividade da MPO, mas nÃo MDA e catalase foram significativamente afetados pelo prÃ-tratamento com ORCL. As observaÃÃes histolÃgicas indicam uma proteÃÃo parcial do ORCL, como UC-TNBS Ã um modelo crÃnico, talvez houvesse necessidade de uma terapia mais prolongada. No modelo de II-I/R, quarenta e cinco minutos de isquemia seguida de uma hora de reperfusÃo da artÃria mesentÃrica superior causou significante aumento nos nÃveis de MPO, catalase, MDA e nitrito, com uma significante diminuiÃÃo dos grupos sulfidrÃlicos nÃo-protÃicos (NP-SH/GSH) indicando um estresse oxidativo. Estes valores foram sigificamente revertido pelo prÃ-tratamento via oral com ORCL (200 e 400mg/Kg), sugerindo que ORCL anula o estresse oxidativo. Animais prÃ-tratados com ORCL (200 e 400mg/Kg, v.o.) ou AK (100mg/Kg, v.o.), 12 e 2 horas antes da administraÃÃo de 20mg/Kg de indometacina causando toxicidade intestinal, foi capaz de ser evidenciado pela diminuiÃÃo nos nÃveis de MPO e nitrito. Diferente da indometacina, ORCL mas nÃo AK falharam em induzir o aumento signifativo na permeabilidade intestinal. Este efeito do ORCL foi similar ao inibidor seletivo de COX-2, rofecoxibe. Estas observaÃÃes sugerem que ORCL Ã isento de toxicidade intestinal diferente da toxicidade intestinal dos inibidores clÃssicos nÃo seletivos da COX. AlÃm disso, ORCL promove cicatrizaÃÃo de feridas aberta ou com incisÃo em ratos evidenciada pela contraÃÃo e tensÃo da pele. Os dados indicam um potencial antiinflamatÃrio do Ãleo-resina da copaÃba e do seu diterpeno Ãcido kaurenÃico, possivelmente mediado pelos mecanismos antioxidantes e anti-lipoperoxidativo / Copaiba oil-resin from Copaifera langsdorffii (Leguminaceae) is a reputed traditional remedy for the treatment of inflammatory conditions and to promote healing of ulcers and wounds. Previous studies established its anti-inflammatory and gastroprotective properties through animal experimentation. The present study extended these earlier studies to analyse the intestinal anti-inflammatory potential of oil-resin Copaifera langsdorffii (ORCL) and its diterpene constituent, kaurenoic acid (KA) in rat models of ulcerative colitis induced by acetic acid (AA-UC), and trinitribenzene sulfonic acid (TNBS-UC), and in indomethacin -and ischemia-reperfusion-induced intestinal inflammation (IND-II and I/R-II). Further, its wound healing potential was evaluated in rats on open and incision wounds. Rats were pretreated orally (15 hrs and 2 hrs before) or rectally 2 hrs before the induction of colitis with ORCL (200 and 400 mg/kg), KA (50 and 100 mg/kg) or vehicle (1 ml, 2% Tween 80 or 1 ml, 2% DMSO). Colitis was induced by intracolonic instillation of a 2 ml of 4% (v/v) acetic acid solution or TNBS (0.25 ml of 20 mg) and 24 hrs or 72 hrs latter, the colonic mucosa was analysed for the severity of macroscopic colonic damage, the myeloperoxidase and the malondialdehyde levels. In AA-UC model, a marked reduction in Gross damage score and in wet weight/length ratio of colonic tissue were evident in animals pretreated orally or rectally with test substances, as compared to vehicle alone-treated controls. This effect was confirmed biochemically by a significant reduction in colonic myeloperoxidase (MPO) activity, the marker of neutrophilic infiltration and by a marked decrease in malondialdehyde (MDA) level, an indicator of lipoperoxidation. Besides, AA elevated increase in the levels of nitrite and catalase activity in colon tissue was also significantly decreased by ORCL treatment. Furthermore, microscopical examination revealed the diminution of inflammatory cell infiltration, and the submucosal edema in colon segments of rats pretreated with ORCL or KA. In a similar manner, in TNBS-UC, a marked reduction in Gross damage score and in wet weight/length ratio of colonic tissue was evident by ORCL pretreatment (400 mg/kg, p.o. or intra-rectal) at 2, 24 and 48 hrs after intracolonic injection of TNBS. MPO activity but not the MDA and catalase levels were significantly affected by ORCL treatment. Histological observations also indicated only a partial protection by ORCL, suggesting that TNBS-UC being a chronic model, a more prolonged therapy may be needed. In the model of I/R-II, five forty minute of ischemia followed by one hour reperfusion of superior mesenteric artery caused significant elevations of MPO, catalase, MDA and nitrite levels with a significant decrease in non-protein sulfhydryls (NP-SH/ GSH) indicating an oxidative stress. These changes were significantly reversed by oral pretreatment with ORCL (200 and 400 mg/kg), suggesting that ORCL obliterates oxidative stress. Pretreatment of animals with ORCL (200 and 400 mg/kg, p.o.) or KA (100 mg/kg, p.o.), 12 and 2 hrs before the administration of 20 mg/kg indomethacin mitigated the intestinal toxicity as evidenced by decreases in tissue levels of MPO and nitrite. Unlike indomethacin, ORCL but not KA at either dose failed to induce a significant increase in intestinal permeability. This effect of ORCL simulated that of a selective COX-2 inhibitor, rofecoxib. These observations suggest that ORCL is devoid of intestinal toxicity unlike the classical non-selective COX inhibitors. Also, ORCL promoted wound healing in rats on experimental open or incision wounds as evidenced by an early wound contraction and increased wound tensile strength. The data indicate a significant anti-inflammatory potential of copaiba oil-resin and its diterpenoid, kaurenoic acid possibly mediated through an antioxidant/anti-lipoperoxidative mechanism(s)

Honey has been shown to have bioactivity. Fynbos (FB) honey was investigated for its bioactivity as this vegetation type is from a unique bio diverse region in the Cape Floristic Kingdom. Six FB and one medical grade Manuka (MAN) UMF 15+ honeys that were of quality grade (Codex Alimentarius) were used. Each honey sample was subjected to in vitro simulated gastro-duodenal digestion and the antioxidant and anti-inflammatory activity of each fraction was determined. These fractions were undigested/raw honey (UD), gastric digest (GD) and gastro-duodenal digest (GDD). Included were pH and digestive enzyme controls. The total polyphenol and the flavonoid content (TPC and TFC) were determined with the Folin-Ciocalteu (F-C) and aluminium chloride methods respectively. Antioxidant activity was measured with the trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. Cellular antioxidant activity (CAA) in the Caco-2 and SC-1 cell lines using the dichloroflourescein diacetate (DCFH DA) assay was investigated. Nitric oxide (NO) scavenging activity was determined with the sodium nitroprusside (SNP) assay. Pro-inflammatory and anti-inflammatory effects of honey were evaluated in non-stimulated and stimulated with LPS/IFN γ murine macrophage RAW 264.7 cells, respectively. Cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was done. TPC and TFC of MAN were higher than that of FB honeys. With GD, TPC and TFC of MAN increased and following GDD, TPC decreased and TFC remained unchanged. In contrast TPC and TFC of FB honey were maintained with GD and GDD. TEAC assay revealed activity by MAN being higher than that of FB honeys. With GD digestion, the antioxidant activity of MAN was unchanged but following GDD, activity was reduced. For FB honeys, TEAC was maintained with GD and GDD. ORAC assay revealed that the activity of MAN was similar to that of FB. Digestion had no effect on activity of both MAN and FB honeys. CAA in the Caco-2 and SC-1 cell line was higher for MAN compared to FB honey. In both cell lines a similar trend was observed where with GD, CAA was unchanged while with GDD, CAA was reduced. This loss of CAA following GDD was found to be due to H2O2 formation as a result of polyphenol degradation in an alkaline environment containing sodium bicarbonate and pancreatin. NO scavenging activity of MAN was greater than FB. For both types of honey with GD, NO scavenging activity was unchanged and with GDD for MAN was reduced and for FB unchanged. Digestion showed an increased pro-inflammatory effect for MAN, FB1, FB2 and FB3. The UD fractions of MAN, FB1 and FB6 had anti-inflammatory effects. FB5 and FB6 honeys showed increased anti-inflammatory activity after GD and GDD. All honey fractions did not show any cytotoxicity. In conclusion, FB honey has antioxidant, pro- and anti-inflammatory properties. With digestion, GD activity was either increased or unchanged while with GDD activity was reduced, lost or unchanged. Observed effects were either due to pH and/or digestive enzyme activity. FB honey with its shown bioactivity could be an important local nutraceutical product. / Dissertation (MSc)--University of Pretoria, 2017. / Anatomy / MSc / Unrestricted

Philosophiae Doctor - PhD / The skin is the largest and most readily accessible organ of the body. The stratum corneum forms the outermost layer of the skin, which functions as a barrier to the external environment and regulates the passage of molecules across the skin. Drug delivery across the skin offers advantages over other routes of administration, bypassing the hepatic first pass metabolism, maintaining therapeutically effective drug levels, and improved patient compliance. Currently, there is a strong trend in the use of plant materials and extracts for medicinal purposes. Devil’s Claw (Harpagophytum procumbens) is a medicinal plant located in many regions throughout Southern and South Africa. It is used for its anti-inflammatory and analgesic properties with the harpagoside and harpagide components present reported to be responsible for these properties. Its activity has been reported to be as a result of the direct and indirect inhibition of Cyclooxygenase- 2 enzyme (COX-2). Efforts to improve the permeation of synthetic conventional compounds are constantly investigated, and great improvements of these formulations have led to very effective formulation of transdermal dosage forms such as anti-inflammatory drugs. The same should be possible for medicinal plants. Optimal permeation across the skin requires a drug to possess lipophilic as well as hydrophilic properties. Research suggests that a drug should have an aqueous solubility of more than 1 mg/ml and an octanol-water partition coefficient (log P) between 1 and 2 to optimally penetrate the skin (higher values indicating increased lipophilicity). Compounds not possessing these characteristics may be facilitated across the skin by the introduction of permeation enhancers in the formulation that include Azone® and sodium dodecyl sulphate xvi (SDS). The main aims of this study were to make an extract of Harpagophytum procumbens and to determine the direct COX-2 inhibition activity of this extract and further to formulate this extract into a pharmaceutical gel, with permeation enhancers that maintains its COX-2 enzyme inhibition qualities after transdermal penetration. The analytical techniques verified the existence of a harpagide and harpagoside components in the crude Harpagophytum procumbens extract and they were quantified in the extract at 3% and 1% respectively. COX-2 inhibition by the Harpagophytum procumbens extract was determined by a direct enzyme inhibition study and quantified by means of measuring the production of the product formed by the enzyme over a time interval in the presence of excess enzyme substrate. Crude Harpagophytum procumbens extract demonstrated a greater COX-2 enzyme inhibition than pure harpagide and harpagoside individually and combined. This indicated the existence of compounds in the extract contributing to this synergistic effect. This was reflected in the IC50 values indicating that the Harpagophytum procumbens crude extract had the lowest IC50 values concentration when compared to the harpagide and harpagoside. Octanol-PBS partition coefficient (log D) experiments were performed with various permeation enhancers and gels with varying combinations in order to determine the changing partitioning properties of harpagide and harpagoside. The octanol-PBS partition coefficient (log D) experiments indicated that harpagoside had a higher log D value than harpagide. The addition of permeation enhancers resulted in changes in the partition co-efficient of the marker compounds. The permeation enhancer, Azone®, resulted in the smallest reduction of Log D for harpagoside and the largest increase in Log D compared to other permeation enhancers tested for harpagide. xvii When the 2 marker compounds were formulated into different gel formulations (hydroxypropyl cellulose, Carbopol Ultrez 21® and Pluronic®), harpagide had the largest increase in Log D with the Carbopol Ultrez 21® gel. Harpagoside had the largest increase in the hydroxypropyl cellulose gel.Combinations of gel formulations with permeation enhancers were tested. The 2 best performing permeation enhancers in hydroxypropyl cellulose gel were SDS and Azone®. These compounds resulted in the largest increase in lipophilic partitioning for harpagide and harpagoside compound and higher values were achieved with SDS. Following the partition co-efficient determination, permeation studies with synthetic membranes were performed with the selected enhancer/gel formulations using Franz diffusional cells. The permeation (flux) across the hydrophobic synthetic membrane (Sil-Tec®) indicated flux of 27.1μg.cm-2.h-1 for harpagoside and 156.0 μg.cm-2.h-1 for harpagide in the hydroxypropyl cellulose gel. Incorporation of Azone® in the hydroxypropyl cellulose formulation resulted in an enhancement ratio (ER) of 14 for harpagoside. The hydroxypropyl cellulose gel with the permeation enhancers (Azone® and SDS) did not result in enhancement of permeation of the harpagide. Permeation of harpagoside and harpagide across the hydrophilic synthetic membrane (Tuffyrn®) yielded flux values of 537.1 μg.cm-2.h-1 and 101.1μg.cm-2.h-1 respectively in the Gel formulations. The Gel formulation containing Azone® and SDS, resulted in similar flux across this membrane compared to the formulation consisting of the Gel only, with the Azone® containing formulation resulting in lower flux for harpagide. Transdermal permeation was measured through excised female human abdominal skin using Franz diffusion cells. xviii Flux values for harpagoside and harpagide were 18.4 μg.cm-2.h-1 and 5.0 μg.cm-2.h-1 respectively when the crude extract was in the hydroxypropyl cellulose gel tested on human skin. Formulation of the crude extract in hydroxypropyl cellulose gel including Azone® and SDS permeation enhancers resulted in enhancement of 8 and 7 respectively for harpagide. Azone® increased the flux of harpagoside 14 times in the same hydroxypropyl cellulose gel formulation. The SDS had very little effect. The enhancement ratios achieved with the human skin was higher than that of the synthetic membranes. Post transdermal COX-2 inhibition activity studies were performed with the Azone® Gel formulation after it permeated across the human skin membrane to determine to what extent inhibitory activity of the crude extract was maintained. The crude drug retained its direct COX-2 inhibitory activity after permeation across the human skin with 77% inhibitory activity.

The widespread acceptance of topical formulations intended for local and/or regional activity has prompted renewed interest in developing a model to determine the bioavailability of drugs in order to establish bioequivalence as a means of evaluating formulation performance of multisource products and also for use during formulation development. Current in vivo techniques such as blister suction and skin biopsy amongst others used to determine the bioavailability and/or bioequivalence of topical formulations are either too invasive to generate appropriate concentration-time profiles or require large numbers of study subjects thereby making the study expensive and time-consuming. Moreover, there are currently no sampling techniques that can demonstrate dermal bioavailability and/or bioequivalence of topical formulations intended for local and/or regional activity. Dermal microdialysis is a relatively new application of microdialysis that permits continuous monitoring of endogenous and/or exogenous solutes in the interstitial fluid. The technique is involves the implantation of semi-permeable membranes which are perfused with an isotonic medium at extremely slow flow rates and collection of microlitre sample volumes containing diffused drugs. Tape stripping, a relatively older technique, has been extensively used in comparative bioavailability studies of various topical formulations. However, due to shortcomings arising from reproducibility and inter-subject variation amongst others, the published FDA guidance outlining the initial protocol was subsequently withdrawn. The incorporation of transepidermal water loss with tape stripping has garnered renewed interest and has been used for the determination of drug bioavailability from a number of topical formulations. Hence the primary objective of this research is to develop and evaluate microdialysis sampling and tape stripping techniques, including the incorporation of the determination of transepidermal water loss, to assess the dermal bioavailability of ketoprofen from topical gel formulations and to develop models for bioequivalence assessment. A rapid UPLC-MS/MS method with requisite sensitivity for the analysis of samples generated from dermal microdialysis was developed and validated which accommodated the microlitre sample volumes collected. An HPLC-UV method was developed and validated for the analysis of samples generated from the in vitro microdialysis and in vivo tape stripping studies. The work presented herein contributes to a growing body of scientific knowledge seeking to develop a model for the determination of bioequivalence of pharmaceutically equivalent topical formulations intended for local and/or regional activity in human subjects.

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. / Land and Food Systems, Faculty of / Graduate

M.Tech. / Purpose: Posterior mechanical neck pain is considered a debilitating musculoskeletal problem and is one of the most common reasons for visiting an emergency department (Murphy, 2000). Anti-inflammatory creams and gels are readily available to individuals suffering from musculoskeletal pain. The purpose of this study was to determine the effectiveness of Traumeel®S ointment together with chiropractic spinal manipulative therapy as a treatment form for posterior mechanical neck pain, with regards to pain, disability and cervical spine range of motion. Method: This study was a comparative study and consisted of two groups of fifteen. The participants were between the ages of eighteen and forty-five, with a half male to female ratio. The potential participants were examined and accepted according to the inclusion and exclusion criteria. Group A received chiropractic spinal manipulative therapy followed by the application of aqueous cream over the upper trapezius muscle area of the posterior neck. This was the placebo group. Group B received chiropractic spinal manipulative therapy followed by the application of Traumeel®S ointment over the upper trapezius muscle area of the posterior neck. This was the experimental group. Objective and subjective findings were based on the above treatment protocols. Procedure: Treatment consisted of seven consultations over a three week period. Objective and subjective readings were taken at the beginning of the first, fourth and seventh consultations. Subjective readings were taken from the Vernon-Mior Neck Pain and Disability Index as well as from the Visual Analogue Scale (VAS). Objective readings were taken from measurements taken from the Cervical Range of Motion device (CROM). At the first to the sixth consultations participants received spinal manipulative therapy with either the application of aqueous cream or the application of Traumeel®S ointment depending on whether they were in group A or group B. Results: Clinically and statistically significant improvements in both group A and group B were seen over the course of the study with regards to cervical spine range of motion, pain and disability. Conclusion: The results show that both treatment protocols were effective in decreasing cervical spine pain and disability and increasing cervical spine ROM. Group B receiving Traumeel®S ointment did show a greater increase in cervical spine ROM clinically, but statistically there was no significant difference between the two groups.

The present program of research, therefore, was designed to attempt to gain new insights into the possible etiology of inflammatory states - for when new information concerning the etiology of these diseases states becomes available, more meaningful screening procedures for anti-inflammatory drugs will certainly be devised. Two preliminary studies have been published.