A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree of Master of Science. Johannesburg, 2014. / Aromatic plants and oils have been used for thousands of years in perfumes, and cosmetics and for their culinary and medicinal purposes. The essential oil from Centella Asiatica growing specifically in South Africa has many therapeutic uses and is used to treat various diseases. Different extraction methods were used on the leaves of Centella Asiatica and the composition of each sample of oil obtained was analysed to see how the composition of each sample is affected. The methods of extracting were optimised specifically for Centella Asiatica. The different extraction methods used were steam distillation, water distillation, solvent extraction and soxhlet extraction. Steam and water distillation were preformed with three different apparatus to compare the efficiency of the extraction and the affect on composition of the oil. It was found that steam distillation using the apparatus described by the British Pharmacopedia Volume IV was the most sufficient apparatus to use to obtain the greatest yield of oil. Soxhlet extraction was found to be the worst extraction method for volatile compounds The optimised parameters for extraction of essential oils from Centella Asiatica using this apparatus were 100 g of leaves at a distillation rate of 2/3 ml min-1 for 75 minutes using 0.4 ml of xylene initially. It was also necessary to perform a 30 minute initial distillation with no plant matter. Steam distillation with this apparatus was found to yield the best quality oil. The major constituents that were found in all the methods were α-carophyllene, carophyllene and germacrene D. There were some similarities found in the compositions of the essential oil between extraction methods in terms of the constituents found. However the abundance of those constituents varied between extractions. Each constituent has a different therapeutic effect. Therefore if an extraction method were to be chosen to extract a some specific constituent like germacrene D and α-carophyllene, steam distillation with the apparatus described by the British Pharmacopedia should be used. However if an extraction method were to be used to extract carophyllene, water distillation should be used. The essential oil extracted using steam distillation yielded a greater amount sesquiterpenoid hydrocarbons. However monoterpenoid hydrocarbons were present in greater amount in the essential oil extracted using water distillation. In the essential oil extracted from Centella Asiatica, 43 constituents were identified from steam distillation extraction representing 98.60% of the composition of oil and 54 constituents representing 98.29% from water distillation extraction. It was found that from steam distillation using fresh leaves compared to dry leaves extraction a greater number of constituents were identified. Salting out was also used for extraction and compared to water distillation and it was found that the salting-out extractions yielded a greater amount of oxygenated polar compounds. A commercial oil of Centella was also analysed and compared to the natural oil. It was found that the commercial oil was a synthetic oil and its composition was completely different from the natural oil and therefore synthetic oils cannot be used therapeutically for substitutes for natural essential oils. Centella Asiatica prefers to grow in damp environments, therefore they are extremely prone to pollution. This was found to also affect the chemical composition of the oil obtained since the soil quality of where the plants were growing was important. This was investigated by spiking the soil of some Centella asiatica plants with chromium(VI), mercury(II) and lead(II). It was found that Centella Asiatica can store heavy metals in the leaves. Since it is a medical plant with many therapeutic uses, this is causes great concern about heavy metal contamination of herbal raw materials of Centella Asiatica. This also highlights the importance of good quality control on Centella Asiatica, so that heavy metals are not ingested. The people in the rural areas who use it as a raw plant for herbal preparations could be at risk of ingesting heavy metals if grown in a polluted area.

Mémoire (M.P.M.O)-- Université du Québec à Chicoutimi, 1991. / Bibliogr.: f. [113]-118. Document électronique également accessible en format PDF. CaQCU

The ability of essential oils (EO) to manipulate the intestinal microbiota may potentiate their application in food as nutraceutical and as prophylactic agents for colonic disease. Little is known about the influence of EO on gut bacteria, the mechanism of their antibacterial action and genotoxicity to the host. Here, the antibacterial activities of EO in pure and in a mixed faecal culture were investigated. These antibacterial activities were further studied to compare the selective nature of EO and their effects on membrane integrity. The growth of gut pathogens and commensals was inhibited in a dose-dependent manner in pure culture, with most of the pathogens, Escherichia coli O157:H7, Clostridium difficile, C. perfringens and Salmonella typhimurium are sensitive to nerolidol, thymol, eugenol and geraniol at a half maximal inhibitory concentration (IC50) of 50-500 ppm. These concentrations of EO and mainly nerolidol were also inhibitory to some gut commensals, in particular affecting Faecalibacterium prausnitzii adversely in pure culture. In contrast, in the mixed culture system beneficial groups of bacteria, including F. prausnitzii, as determined by qPCR of 16S rRNA genes were not affected. Thymol and geraniol at 500 ppm suppressed the growth of total bacteria, resulting in minimal fermentation. A lower dose of 100 ppm of EO compounds was effective in suppressing the pathogen, C. difficile with no concern for commensal bacteria or their fermentation products, acetate, propionate and butyrate. This study also discovered that the proteome of commensal, Faecalibacterium prausnitzii and pathogenic gut bacteria, Escherichia coli, in response to EO compounds are affected differently. Thymol and eugenol down-regulated virulence factors in E. coli. The tested EO compounds were not genotoxic in the comet assay at non-toxic doses. Differential effects of EO compounds on gut pathogens and commensals and their non-toxicity but geno-protective properties could be applicable in improving gut health in man.

Protium é o principal gênero pertencente à família Burseraceae e um dos gêneros mais comum na América do Sul, sendo representativo na flora da Região Amazônica. Na medicina popular, as oleorresinas das espécies Protium são amplamente utilizadas para diversos propósitos pelas tribos nativas das regiões onde estas espécies ocorrem. No presente estudo, os óleos essenciais de quatro espécies Protium foram extraídos, quantitativamente por hidrodestilação. Estas resinas foram coletadas de árvores catalogadas no Instituto Nacional de Pesquisas da Amazônia (INPA), na Reserva Ducke, Manaus/AM, em novembro de 2004. Foram determinadas as características físico-químicas destes óleos. Técnicas espectroscópicas, como espectrometria de absorção no infravermelho (IV), espectrometria de absorção no ultravioleta (UV) e espectrometria de massas (EM), foram utilizadas como ferramentas qualitativas nas análises destes óleos. Foi utilizada cromatografia em camada delgada (CCD) para a separação dos constituintes destes óleos em frações. Para identificação e quantificação dos componentes destes óleos foi utilizada cromatografia gasosa acoplada a espectrometria de massas (CG/EM). Os espectros de massas obtidos para os componentes individuais dos óleos foram analisados por comparação com os espectros da literatura [52]. Os rendimentos nas extrações foram: 3,1% (P. hebetatum), 3,33% (P. nitidifolium), 2,6% (P.divarictium) e 2,77% (P.amazonicum). No óleo extraído da espécie P. hebetatum foram identificados 17 compostos, sendo os monoterpenos constituintes predominantes na composição deste óleo. No óleo extraído das espécies P. nitidifolium e P. divarictium foram identificados 21 e 23 componentes, respectivamente. No óleo extraído da espécie P. amazonicum foram identificados 18 compostos. Uma característica notável nesta amostra é a predominância de sesquiterpenos em sua composição. A literatura relata a predominância de sesquiterpenos exclusivamente em óleos essenciais extraídos de folhas de espécies Protium spp. É importante salientar ainda, que não há relatos na literatura acerca de óleos essências das espécies P. amazonicum e P. divarictium. / Protium is the main genus in the Burseracea family and is one of the most widespread genera in South America, being very representative in the flora of the Amazon region. In the folk medicine, the oleoresins from species of Protium are widely used for several purposes by the native tribes in the regions where they occur. In the present study the essential oils of four species of Protium were extracted quantitatively by hidrodestillation from aged resins. These samples were collected from trees catalogued by the National Institute of Resesarch of Amazonia (INPA), in the National Forest Ducke, Manaus – Amazonas, in November 2004. The oils extracted were analyzed in order to determine its physical-chemical characteristics. Spectroscopic techniques such as infrared absorption spectroscopy, ultraviolet absorption spectroscopy and mass spectrometry were used to analyze these oils. Thin layer chromatography was used to separate the fractions of the components from these samples. For the identification and quantification of the components, the coupled gas chromatography-mass spectrometry technique was employed. The registered mass spectra of these oils compounds were compared with those of the literature [52]. The extractions yields were 3.1% (P. hebetatum), 3.33% (P. nitidifolium), 2.6% (P.divarictium) and 2.77% (P.amazonicum). In the P. hebetatum oil 17 compounds were identified, being the monoterpenes predominate in this oil composition. In the P. nitidifolium oil 21 compounds were identified and, in the P. divarictium oil 23 compounds. In the P. amazonicum oil 18 compounds were identified, and the major constituent identified was the sesquiterpene cedrane. A noticeable feature is the predominance of sesquiterpenes in the composition of the P. amazonicum oil. These compounds are reported to be majoritary in essential oils extracted of Protium species leaves. It is important to notice that, in the present moment, no data was reported about P. amazonicum and P.divarictium essential oils. The results show a high potential of these species as a relevant research area, mainly in Brazil that owns the major flora of the world, the Amazon Forest.

Esse estudo é um estudo teórico, baseado em revisão bibliográfica. Ele objetivou a construção de um panorama conceitual da aromaterapia, considerandose sua história e desenvolvimento, além de propor um modelo integrativo dos aspectos fisiológicos e psicossociais com base nas premissas psiconeuroendocrinoimunológicas. Aromaterapia é uma prática milenar, que passou por diversas mudanças ao longo da história e por esse motivo, atualmente, apresentase conceitualmente confusa e imprecisa. Seu ressurgimento nos anos 30 permitiu um início da visão científica do assunto, que, no entanto, evoluiu lentamente pelas dificuldades metodológicas encontradas. A organização do panorama atual dessa terapia permitiu observar que existem muitos países que estudam a aromaterapia, no entanto, com abordagens e visões distintas, de modo que tornase complexa a intersecção dos estudos. No seu estudo científico diversos elementos devem ser aqui considerados, como tipo de estudo (teórico, préclínico ou clínico), variáveis a serem controladas (farmacológicas, dos sujeitos e de procedimento) e questões abordadas (quanto ao efeito dos óleos essenciais, quanto aos seus mecanismos de ação e quanto à influência das variáveis em ambos). Além disso, atualmente existem diversas abordagens: filosófica (baseada em filosofias de saúde orientais), psicológica (baseada no conceito de memória olfativa), farmacoquímica (baseada em farmacologia e química dos óleos essenciais), neurológica (baseada nas neurociências) e psiconeuroendocrinoimunológica (baseada na psiconeuroendocrinoimunologia). Esse trabalho se focou nos estudos préclínicos e clínicos de aromaterapia, a partir de uma visão psiconeuroendocrinoimunológica. A fim de servir como um passo inicial à padronização científica do assunto, foi proposto uma definição mais objetiva de aromaterapia, a partir da qual o trabalho foi desenvolvido. Dentro do modelo psiconeuroendocrinoimunológico, a aromaterapia pode ter efeitos diretos ou indiretos nos sistemas nervoso, endócrino, imune e psicológico, sendo esses efeitos tanto fisiológicos quanto psicológicos e dados por mecanismos de ação farmacológicos e olfativos. Os aromas sempre têm efeitos farmacológicos, independente da via de aplicação utilizada, no entanto, quando se utiliza a via inalatória, são acrescidos a esses efeitos farmacológicos os efeitos olfativos, que são próprios do sistema olfativo e diferenciados. O estudo do olfato é indispensável para o entendimento científico da aromaterapia e ele tem se desenvolvido amplamente, apesar de que ainda existem muitos elementos a serem esclarecidos. Com isso, os estudos na área da aromaterapia científica tem evoluído cada vez mais, permitindo estudos mais minuciosos e conclusivos a respeito do funcionamento dos óleos essenciais no organismo e na mente. Um caminho pra esses estudos, dentro do modelo psiconeuroendocrinoimunológico, é o estudo da relação entre aromaterapia e stress. A premissa básica da teoria de stress é estudar a integração do corpo e da psique. Esse estudo concluiu que a psiconeuroendocrinoimunologia é um modelo útil para estudar a aromaterapia, por permitir o seu estudo científico integrando seus efeitos fisiológicos e psicológicos, e que a organização realizada permitiu uma fundamentação teórica para a elaboração, em futuros projetos na área, de métodos científicos em aromaterapia, stress e psiconeuroendocrinoimunologia. / This is a theoretical study, based on bibliographical revision. It aimed to build a conceptual panorama of aromatherapy, considering it\'s history and development, and also propose an integrative model of the physiological and psychosocial aspects, based on the psychoneuroendocrineimmunological premisses. Aromatherapy is a millenarian practice, that suffered many changes throughout history, and, for this reason, nowadays, it is confusing and uncertain. It\'s reappearance in the 30\'s permitted a start of the scientific view of the theme. However, the scientific view evolved slowly because of the methodological difficulties that were found. The organization of the current panorama of this therapy permitted observing that many countries study aromatherapy. However, there are many different approaches and views of the subject, in a way that it\'s complex to do the intersection of the studies. In it\'s scientific study there are many elements to be considered, such as type of study (theoretical, preclinical or clinical), variables to control pharmacological, subject and procedural) and questions studied (about the effects of essential oils, about their action mechanisms and about the influence of the variables on both). Furthermore, currently there are many approaches to the study: philosophical (based on oriental heath philosophy), psychological (based on the concept of olfactory memory), pharmacochemical (based of pharmacology and essential oil chemistry), neurological (based on neuroscience) and psychoneuroendocrineimmunological (based on psychoneuroendocrineimmunology). This work focused on preclinical and clinical studies, from a psychoneuroendocrineimmuneological point of view. In order to serve as a first step to the scientific standardizing of the subject, a more objective definition of aromatherapy was proposed, from which the study developed the subject. In the psychoneuroendocrineimmunological model, aromatherapy may have direct or indirect effects in the nervous, endocrine, immune and psychological systems. These effects can be physiological and psychological and they are caused by pharmacological and olfactory mechanisms. Aromas always have pharmacological effects, independently from application via, but when the inhalation is used the olfactory effects are added to these pharmacological effects. The olfactory effects are different from the others and characteristic of the olfactory system. The study of olfaction is indispensable to the scientific understanding of aromatherapy and it has evolved immensely, although there are still many elements still to be understood. Consequently, the studies in aromatherapy also have evolved more, permitting more minute and conclusive studies about the functioning of essential oils in the organism and mind. A path to this kind of study, in the psychoneuroendocrineimmunological model, is the study of the interaction between aromatherapy and stress. The basic premiss of the theory of stress is to study the integration of body and mind. This study concluded that psychoneuroendocrineimmunology is a useful model to study aromatherapy because it permits the scientific evaluation of both physiological and psychological effects of aromatherapy. It also concluded that the organization of the current panorama permitted a theoretical foundation for elaboration of scientific methods in aromatherapy, stress and psychoneuroendocrineimmunology in future studies.

L’usage de la phytothérapie pour la prévention de pathologies récurrentes dans les élevages s’est récemment développé. Les huiles essentielles (HE) sont des extraits végétaux complexes, constituant une source d’alternatives de par leur composition chimique variée et d’intéressantes activités biologiques. Ce projet de thèse a été réalisé dans un contexte industriel, avec pour objectif principal la mise en évidence des propriétés des HE et de leurs mélanges par des méthodes in vitro. Les mélanges formés doivent permettre de cumuler les activités naturelles des HE dans les sphères respiratoires, digestives et immunitaires et dans la prévention des mammites. Une sélection en entonnoir intervient d’après une trame d’essais spécifiques à chaque sphère. L’HE de girofle, intégrée dans les mélanges, développe d’après différentes méthodes biochimiques de fortes activités antioxydantes et anti-inflammatoires. Les HE de cannelle et d’origan se sont illustrées pour leur capacité à inhiber le développement des pathogènes comme les bactéries sous forme planctonique ou de biofilm, les virus ou les parasites internes. Les effets sur le biofilm bactérien ont notamment été observés par microscopie confocale à balayage laser. Enfin la capacité de l’HE de girofle à moduler la réponse inflammatoire a été démontrée sur le modèle de macrophage RAW264.7. La phagocytose, l’expression de la nitroxyde synthase et la sécrétion d’interleukine-6 ont été suivis par criblage à haut débit associé à la microscopie à épifluorescence. La caractérisation des mélanges a abouti à la formulation des noyaux Respilor®, Digelor®, Immulor® et Mastilor®, testés in vivo et commercialisés par BioArmor. / The use of phytotherapy for the prevention of livestock associated pathologies is increasing since recent years. Essential oils (EO) are complex plant extracts, forming a large source of alternatives due to their chemical diversity and interesting biological activities. This project, carried out over three years and anchored in an industrial context, aims to highlight the properties of EO and their blends using in vitro assays. The objective is to cumulate the natural properties of EO through their blending. The prevention of respiratory and digestive tract diseases, of mastitis and the promotion of the animals’ immunity are particularly targeted. A funnel selection takes place according to tests selected for their specificity to each area. Clove EO, integrated in the mixtures, develops according to different biochemical methods strong antioxidant and anti-inflammatory activities. EOs of cinnamon and oregano have been shown to inhibit the development of pathogens such as planktonic or biofilm embedded bacteria, viruses or internal parasites. The effects on the bacterial biofilm were especially measured using confocal laser scanning microscopy. Finally, the ability of clove EO to modulate the inflammatory response has been demonstrated on the RAW264.7 macrophage cells. Phagocytosis, the expression of Nitroxide synthase, and the secretion of interleukin-6 were followed by high content screening methods associated with epifluorescence microscopy. The characterization of the mixtures resulted in the formulation Respilor®, Digelor®, Immulor® and Mastilor®, four blends of EO tested in vivo and commercialized by BioArmor.

“Make it essential” es el nombre de nuestro proyecto de negocio que plantea poner fin a una necesidad y problemática que permanece en la sociedad y que, en los últimos años, se ha incrementado de manera significativa en los sectores de educación y laboral como es el estrés, a través de una solución innovadora que les permita llevar una vida más plena. El proyecto se basa en la venta de joyas difusoras de aceites esenciales que cumplen el propósito de aliviar el estrés que es originado por los estudios, el trabajo y también problemas por los que pueda estar pasando la persona. A lo largo del ciclo, hemos logrado validar la idea de negocio a través del planteamiento de hipótesis y la ejecución del Concierge que fue impulsado por las estrategias de marketing en redes sociales. El público logró tener una buena aceptación del producto gracias al valor diferenciado que posee y obtuvo ventas por 212 unidades a la fecha, que están valorizadas en S/. 12,321. Asimismo, la empresa tiene como objetivo crecer con el tiempo, tanto en ventas como en capacidad instalada, con la finalidad de lograr atender la demanda proyectada y expandirse a otros territorios. Es decir, el análisis de viabilidad en un horizonte de 5 años nos indica que el proyecto está valorizado en S/. 47,963 gracias a la proyección de flujos de efectivo traídos a valor presente, lo cual nos indica que el proyecto es viable y sostenible en el tiempo. / "Make it essential" is the name of our business project that proposes to end a need and problem that remains in society and that in recent years has increased significantly in the education and work sectors such as stress, through an innovative solution that allows them to lead a fuller life. The project is based on the sale of jewelry diffusers of essential oils that fulfill the purpose of relieving the stress that is caused by studies, work and also problems that the person may be going through. Throughout the cycle we have managed to validate the business idea through the hypothesis approach and the execution of the Concierge that was driven by social media marketing strategies. The audience managed to have a good acceptance of the product thanks to the differentiated value that it has, achieving sales of 212 units to date that are valued in S /. 12,321. The company aims to grow over time, both in sales and installed capacity to meet projected demand and expand to other territories. The feasibility analysis over a 5-year horizon tells us that the project is valued in S/. 47,963 thanks to the projection of cash flows brought to present value, which tells us that the project is viable and sustainable over time. / Trabajo de investigación

Although the antomicrobial nature of essential oils and their major constituents, the terpenoids, has been widely investigated the mechanism of their antimicrobial action has not been subject to the same scrutiny. In this study the membrane disruptive nature of the terpenoids has been determined by experiments on the effects of terpenes on both microbial membrane and model lipid bilayer systems. These terpenes exhibited a range of membrane damaging effects. Experiments showed that the terpenoids were able to increase disorder in DPPC bilayers, and that antimicrobially active terpenoids cause increased membrane permeability in living cells. The effect of molecular structure on antimicrobial activity and activity differences between each organism were determined for 60 terpenoids. Terpenoids with a low water solubility were found to be inactive. Hydrogen bond donor capacity and hydrogen bond acceptor capacity were found to be the molecular parameters which most strongly discriminated between activities against individual organisms. Activity against Gram negative Escherichia coli and Pseudomonas aeruginosa was associated also with a molecular size parameter. The protective nature of the outer membrane of P. aeruginosa and E. coli was also investigated. The evidence presented in this thesis has enabled the postulation of a two stage process to explain the overall mode of action of these compounds. / Doctor of Philosophy (PhD)

Logging and forest thinning operations generate large amounts of residues in the form of small trees, branches and foliage that are usually left on the ground to decay or are burned. These resources are readily available, but it is uneconomical to remove them from the forest. The potential utilization of these resources was investigated for western juniper. This species is invading rangeland in the Interior West, altering the ecology of the land, and farmers are being encouraged to remove it. However, limited markets for the wood make removal costly. Developing uses for other parts of the stem could make removal more economically attractive. Western juniper foliage and twigs were steam distilled to obtain essential oils which were tested for their activity against subterranean termites and fungi. Residues from the steam distillation were pretreated with dilute sulfuric acid and digested with enzymes to determine their digestibility. The goal was to determine if an integrated operation involving steam distillation to remove essential oils, followed by enzymatic digestion of extraction residues was feasible. Essential oil recovery from foliage averaged 0.77% wet weight, while yields from twigs were about 0.13% wet weight. Foliage essential oil showed high antifungal and termiticidal activities at the concentrations tested. Total sugar yields from foliage extraction residues (40-55%) were higher than those obtained from branchwood residues (15-25%). The highest yields were obtained at 121°C and 1 hour pretreatment in 1% sulfuric acid. These results show that an integrated operation involving steam distillation of western juniper foliage to remove essential oil and enzymatic digestion of extraction residues is technically feasible. Additional work to determine the optimum distillation and pretreatment conditions for foliage would be needed. / Graduation date: 2012

Thesis (M. Sc.(Agric))(Horticulture)-University of Pretoria, 2005. / Includes bibliographical references. Available on the Internet via the World Wide Web.