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.

Cette thèse s'inscrit dans le cadre de la thématique du Laboratoire de Pharmacognosie de l'UFR des Sciences de santé, au sein de l'Université de Bourgogne Franche-Comté et du Laboratoire de Produits Naturels de la Faculté de Sciences, au sein de l'Universidad de Los Andes (Venezuela). Elle vise essentiellement l'isolement, purification et identification de molécules d'origine végétale avec activité biologique. Dans ce contexte, l'étude de plantes vénézuéliennes, Bursera inversa (Burseraceae), Lepechinia Bullata (Lamiaceae) et Hydrocotyle multifida (Araliaceae) avec des cultivars Pittosporum tenuifolium 'Variegatum' et Pittosporum tenuifolium 'Gold star' a conduit à l'obtention de treize glycosides naturels, un dérivé de l'acide caféique et diverses hydrocarbures à longue chaîne par les techniques de chromatographie de couche mince préparative, de chromatographie liquide à moyenne pression, chromatographie liquide haute performance, et de chromatographie liquide sous vide. Les structures ont été élucidées principalement par les techniques spectroscopiques de RMN-1 D et -2D, et de spectrométrie de masse. Il s'agit de treize saponines triterpéniques de type oléanane (parmi lesquelles neuf sont des nouveaux composés naturels) ainsi que de l'acide rosmarinique et des alcanes et esters d'acides gras à longue chaîne. / This thesis was carried out in the Laboratory of Pharmacognosy, on the Health Sciences section of Université de Bourgogne Franche-Comté (France) and also in the Laboratory of Natural Products of Sciences Faculty of Universidad de Los Andes (Venezuela). The aim of this thesis was the isolation, purification and identification of bioactive molecules from several plant species. ln this context, the study of Venezuelan species, Bursera inversa (Bùrseraceae), Lepechinia Bullata (Lamiaceae) and Hydrocotyle multifida (Araliaceae), together with the cultivars, Pittosporum tenuifolium 'Variegatum' and Pittosporum tenuifolium 'Gold star' was carried out. The study led to the isolation of thirteen natural glycosides, one caffeic acid derivative and several long chain hydrocarbons by column chromatography, preparative thin layer chromatography, medium pressure liquid chromatography, high performance chromatography and vacuum liquid chromatography. The structures were elucidate mainly by spectroscopie techniques, NMR-1 D and 2D, and mass spectrometry. The compounds were characterized as thirteen oleanane-type saponins (among them nine are new natural compounds), rosmarinic acid and several long chain alkanes and fattv acid esters / Este trabajo se presenta bajo la forma de secciones, donde en primer lugar se expone una breve introducción acerca de los géneros de interés para este estudio, como son Bursera (Burseraceae), Hydrocotyle (Araliaceae), Lepechinia (Lamiaceae) y Pittosporum (Pittosporaceae); así como los motivos que llevaron a la realización de este estudio fitoquímico. Cada género es tratado en un capítulo diferente, que abarca una revisión bibliográfica extensa (considerando los aspectos etnobotánicos, fitoquímicos y farmacológicos) del género, seguida por el procedimiento experimental llevado a cabo en cada especie analizada y por último el análisis de los resultados obtenidos.Los compuestos aislados de cultivares de Pittosporum tenuifolium estudiados: 'Variegatum' y 'Gold Star' se encuentran descritos en el primer capítulo entre los que se encuentran ocho nuevas saponinas triterpénicas tipo-oleanano: PT-PI y PT-P4 de los tallos deP. tenuifolium 'Variegatum', PT-P2 y PT-P3 de las hojas de este mismo cultivar, PT-P7 y PT-P8 de los tallos de P. tenuifolium 'Gold Star' y por último, PT-P5 y PT-P6 aisladas de los tallos de ambos cultivares. Además también se describe el compuesto ya conocido, udosaponinaF (PT-P9), identificado en los tallos de P. tenuifolium 'Variegatum' De las especie Hydrocotyle multifìda se aislaron cinco saponinas triterpénicas, descritas en el segundo capítulo, las cuales tienen al ácido oleanólico como genina. Dos de las cinco saponinas son productos naturales nuevos, HmE-P3 y HmE-P2 minoritario; las otras tres saponinas son compuestos ya conocidos, momordin I (HmE-P1), sandrosaponina X (HmE-P2 mayoritario) y hemslósido Mal (HmE-P4) En el tercer capítulo que abarca al género Bursera, incluye los resultados del estudio fitoquímico de las semillas de Bursera inversa, entre los que se puede mencionar la identificación de 45 compuestos en las semillas de esta planta. El último capítulo contiene la descripción del compuesto mayoritario, aislado de las mias de Lepechinia bullata e identificado como el ácido rosmarínico (LB-TI).

Ces travaux de thèse concernent l’étude phytochimique de cinq plantes médicinales dont les espèces sélectionnées sont endémiques du Venezuela : Palicourea demissa Standl. et Hydrocotyle umbellata L., mais aussi Bauhinia cumanensis H.B.K, Guazuma ulmifolia Lam. et Urena sinuata L. Vingt-deux composés ont été purifiés et identifiés à partir des différents extraits étudiés ; il s’agit notamment de terpènes, de flavones et de saponines. Six de ces substances naturelles, parmi lesquelles figurent plusieurs saponines de types spirostane et ergostane, sont décrites pour la première fois. / This work is dedicated to the phytochemical study of five medicinal plants whose selected species are endemic of Venezuela: Palicourea demissa Standl., and Hydrocotyle umbellata L., but also Bauhinia cumanensis H.B.K, Guazuma ulmifolia Lam., and Urena sinuata L. Twenty-two compounds were purified and identified from the various studied extracts; they are in particular terpenes, flavones and saponins. Six of these natural products, among which appear several spirostane- and ergostane-types saponins, are described for the first time.