published_or_final_version / abstract / toc / Paediatrics / Master / Master of Philosophy

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Bacterial and fungal pathogens have developed numerous defence mechanisms against antimicrobial chemical agents, and resistance to old and new produced drugs are on the rise. Discovery of natural products derived from plants with diverse chemical structures and novel mechanisms of action to treat these notorious pathogens is a priority. Biotechnology (discussed in Chapter 1) has much to offer as a pharmacological tool and in the general study of medicinal plants. The Genus Salvia (Lamiaceae) has gathered much interest as these plants manufacture a diverse range of secondary metabolites including flavonoids, tannins and terpenoids. Of particular interest are the terpenoids which are largely implicated in the efficacy of Salvia plants as traditional medicines contributing to their pharmacological actions (discussed in Chapter 2). Due to the importance of these plants as herbal remedies, in this study, biotechnological techniques such as tissue culture and Agrobacterium-mediated transformation were applied on Salvia runcinata L.f., a South African medicinal plant, in an attempt to enhance the metabolomic profile and its bioactivity. Like so many other sages, S. runcinata has been used in folk medicine to treat a variety of ailments. Application of biotechnology was viewed as an important value adding platform for this species, assisting with its commercialisation for the cosmeceutical and pharmaceutical industries. Therefore the study had three foci: (1) to determine the seed germination behaviour and optimal conditions for micropropagation; (2) to develop a protocol that would be efficient whilst being simple for genetic transformation; and lastly, (3) to conduct phytochemical studies on in vitro generated S. runcinata transgenic hairy root and in vitro organ cultures by comparing these to glasshouse plants as potential therapeutic sources of natural compounds used in the treatment of infections in plants and humans. Data generated is thus summarised in three research chapters and Chapter 3 describes the formulated procedures assisting with in vitro seed germination and micropropagation of S. runcinata. The efficacy of smoke and scarification treatments for germination improvement was initially tested coupled to the evaluation of different hormonal combinations and different explant types which would aid with inducing adventitious shoot formation in vitro. The most effective germination treatment proved to be a 3 min exposure of seeds to 25% (w/v) H2SO4 combined with a concentration of 10-5 M smoke solution, resulting to more than 80% germination. Shoot proliferation was significantly higher using nodal explants with the addition of 4.43 μM BA. The protocol established in this part of the study is viable for large scale commercial production of S. runcinata as it would yield 1296 to 46656 viable plants in 4 to 6 months from one nodal explant. Micropropagation was applied also as a pre-emptive measure to ease pressure on the wild plants as the demand for S. runcinata is anticipated to increase due to its growing economic value as it is one of two South African sages with epi-α-bisabolol that is sought after by the pharmaceutical and cosmeceutical industries. This makes the protocol developed in this part of the study suitable for ex situ conservation of S. runcinata plantlets. Evaluations on the transgene transfer capacities of two different agropine strains (A4T and LBA 9402) of Agrobacterium rhizogenes to induce hairy root cultures of S. runcinata explants on nodal and leaf explants were conducted (reported in Chapter 4). Hairy roots formed 3 to 4 weeks after inoculation of the explants and these agropine strains showed different abilities for genetic transformation with the LBA 9402 strain producing significantly more roots on each explant compared to the A4T strain (P=0.0075). However, none of the LBA 9402 derived clones and only 2 clones generated through A4T transformation survived subculturing. The polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the presence and transcription (respectively) of rol A, rol B, rol C and ags genes which are mobilised from the transfer-DNA (T-DNA) fragment of the root-inducing (Ri) plasmid of A. rhizogenes to the plant genome during transformation. The two A4T clones, termed here A4T3 and A4T5, were stably transformed, Southern blot analysis using rol A as a probe further validated the integration of one copy of the rol A gene. Transformed hairy roots, untransformed roots from tissue cultured plants, tissue culture-derived plants and glasshouse-grown plants were profiled for secondary metabolites by thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) in Chapter 5. In this part of the study, it is clear that the use of tissue culture as a propagation system did not negatively affect the volatile compound profile of S. runcinata and plants had a similar essential oil content to that reported by Kamatou et al. (2008), leading to a conclusion that in vitro plants maintained their biochemical integrity even under an alternative micro-controlled environment. Similarly to others, Ri-transformation was explored as an avenue to alter secondary metabolism creating inter-clonal variation. Transformed clones were distinguishable, displaying more of some primary metabolites including sucrose, galactose, sorbose and fructose than the leaf extracts. With the current GC-MS methods used, this clear distinction was not obvious at the secondary metabolite level. In general, solvent extracts (acetone and methanol:dichloromethane (MetOH: DCM) (1:1 v/v) exhibited good to moderate antibacterial activity with the minimum inhibitory concentration (MIC) values ranging from 0.39 to 0.78 mg ml-1. However, in vitro plant cultures were the most potent against two Gram-negative bacterial strains: Escherichia coli (ATCC 11775) and Klebsiella pneumoniae (ATCC 13883), and two Gram-positive bacterial strains: Bacillus subtilis (ATCC 6051) and Staphylococcus aureus (ATCC 12600). The hairy root extracts did not show any activity against fungi, Fusarium subglutinans (MRC 0115) and Fusarium proliferatum (MRC 6908). Micropropagation therefore proves to be an interesting avenue for commercial production of S. runcinata, supplying plants with an improved pharmacological activity. Hence the biotechnological approach applied here is a viable strategy for the production of medicinal bioactives from S. runcinata. / AFRIKAANSE OPSOMMING: Bakterieë en fungi patogene het baie verskeie meganismes ontwikkel teen antimikrobiese chemiese agente, en weerstand teen ou en nuwe chemise stowwe is besig om te vergroot. Daarom is dit belangrik om natuurlike plantaardige produkte met diverse chemiese strukture en unieke werkings meganismes te ontdek waarmee hierdie berugte patogene beveg kan word. Biotegnologie (wat in Hoofstuk 1 bespreek word) kan gebruik word as 'n farmakologiese hulpmiddel in die algemene studie van plante. Die Klas (Genus) Salvia (Lamiaceae) het al baie aandag getrek aangesien hierdie plante 'n wye reeks sekondêre metaboliete vervaardig wat flavonoïede, tanniene en terpenoïede insluit. Veral van belang is die terpenoïde wat betrokke is by die doeltreffendheid van die Salvia plante as tradisionele medisyne, aangesien dit bydra tot hulle farmalogiese aksie (wat in Hoofstuk 2 bespreek word). Aangesien hierdie plante sulke belangrike kruie is, word daar in hierdie studie, biotegnologiese tegnieke soos die kweek van weefsel en Agrobacterium-bemiddelde transformasie op Salvia runcinata L.f. toegepas om die metabologiese profiel en die bioaktiwiteit daarvan te verbeter. Soos baie van die salies is S. runcinata tradisioneel dikwels gebruik om allerhande siektetoestande te behandel. Die toepassing van biotegnologie word beskou as 'n belangrike manier om waarde by te voeg sodat hierdie plant kommersieei deur die kosmetiese en farmakeutiese bedrywe gebruik kan word. Daarom is daar op drie dinge gefokus: (1) die ontkiemings gedrag van saad en die optimale toestande vir mikrovoortplanting (2) die ontwikkeling van protokol wat eenvoudig maar doeltreffend is vir genetiese transformasie, en die (3) fito-chemise studies op in vitro genereerde S. runcinata transgeniese harige wortels en in vitro orgaan kwekings deur om hulle te vergelyk met kweekhuis plante as potentiële terapeutiese bronne van natuurlike samestellings vir die behandeling van infeksies in beide plante en mense. Die data wat gegenereer is, is opgesom in drie hoofstukke, en in Hoofstuk 3 word die prosedures wat gebruik word in die in vitro saad ontkieming en die mikro voortplanting van S. runcinata, bespreek. Die doeltreffendheid van rook en skarifikasie behandeling vir die verbetering van ontkieming is eers getoets en gekoppel aan die evaluering van verskillende hormoonkombinasies en verskillende eksplant tipes wat lei tot die formasie van uitloopsels in vitro. Daar is gevind dat die effektiefste behandeling vir ontkieming, 'n 3-minuut blootstelling van saad aan 25% (w/v) H2SO4 gekombineer met 'n konsentrasie 10-5 M rook oplossing is. Dit het gelei tot meer as 80% ontkieming. Daar was baie meer uitloopsels toe nodale eksplante gebruik is met die byvoeging van 4.43 μM BA. Die proktokol wat hier gevestig is, kan op groot skaal gebruik word vir die kommersiële produksie van S. runcinata, want 1296 tot 46656 lewensvatbare plante kan binne 4 ot 6 maande van een nodale eksplant gemaak word. Mikro voortplanting is toegepas as 'n voorkomende maatreel om die druk op die natuur te verminder omdat daar verwag word dat die vraag na S. runcinata sal toeneem na gelang die groeiende ekonomiese waarde daarvan toeneem. Dit is een van twee Suid-Afrikaanse salies met epi-α-bisabolol wat deur die farmakeutiese en die kosmetiese bedrywe gebruik word. Dit beteken dat die protokol wat hier ontwikkel is, geskik is vir die ex situ bewaring van S. runcinata plante. Die transgeen oordrag van twee verskillende agropien tipes (A4T and LBA 9402) van Agrobacterium rhizogenes is geevalueer (en in Hoofstuk 4 beskryf). Harige wortels het 3 tot 4 weke na die inenting van die eksplante gevorm en hierdie agropien tipes het verskillende vermoëns vir genetiese transformasie getoon, met die LBA 9402 tipe wat baie meer wortels op elke eksplant voorgebring het in vergelyking met die A4T tipe (P=0.03116). Geen van die LBA 9402-afgeleide klone en slegs 2 klone wat deur A4T transformasie genereer is, het oorleef. The polimerase ketting reaksie (PCR) en die teenoorgestelde trenskriptasie-polimerase (RT-PCR) ketting reaksie het die teenwoordigheid en transkipsie (onderskeidelik) van rol A, rol B en rol C en ags gene, wat oorgedra word deur die oordrag DNA (T-DNA) fragment van die wortel induserende (Ri) plasmied van A. rhizogenes na die plant genoom tydens transformasie, bevorder. A4T klone, hier A4T3 and A4T5 genoem, is stabiel transformeer. Southern blot ontleding het met die gebruik van rol A, die integrasie van een kopie van die rol A geen, bevestig. In Hoofstuk 5 is transformeerde harige wortels, ongetransformeerde wortels van weefsel gekweekte plante, weefsel gekweekte plante, en kweekhuis plante deur dun-laag chromatografie (TLC) en gas-chromatografie-massa spektrometrie (GC-MS) geprofiel vir sekondêre metaboliete. In hierdie deel van die studie is dit duidelik dat die gebruik van weefsel kwekery as 'n voortplantsisteem nie 'n negatiewe effek gehad het op die vlugtige samestelling profiel van S. runcinata nie en dat plante 'n sootgelyke essentiële olie inhoud het as wat deur Kamatou et al. (2008) bevind is. Dit lei tot die gevolgtrekking dat in vitro plante hulle biochemiese integriteit behou selfs onder alternatiewe mikro-beheerde omgewings. Ri-transformasie is ondersoek as 'n manier om sekondêre metabolisme te verander om interkloon variasie te skep. Getransformeerde klone kon uitgeken word, aangesien dit meer primêre metaboliete soos sukrose, galaktose en fruktose insluit as die blaar ekstrakte. Hierdie verskil was nie met die huidige GC-MS metodes so duidelik sigbaar op die sekondêre metabolitiese vlak nie. Oor die algemeen toon ekstraksie met asetoon en methanol dichlorometaan (MetOH: DCM) (1:1 v/v) goeie tot gemiddelde antibakteriese aktiwiteit met die minimum remmende konsentrasie (MIC) waardes van 0.39 tot 0.78 mg ml-1. Die in vitro plant kulture het egter sterker weerstand gebied teen twee Gram-negatiewe bakteriese tipes: Escherichia coli (ATCC 11775) en Klebsiella pneumoniae (ATCC 13883), en teen twee Gram-positiewe bakteriese tipes: Bacillus subtilis (ATCC 6051) en Staphylococcus aureus (ATCC 12600). Die harige wortel ekstrakte het geen aktiwiteit teen die swamme, Fusarium subglutinans (MRC 0115) en Fusarium proliferatum (MRC 6908) getoon nie. Mikro-voortplanting is dus 'n interessante manier om S. runcinata kommersieel te produseer aangeien die plante verbeterde farmalogiese aktiwiteit toon. Die biotegnologiese benadering wat hier toegepas word, is 'n praktiese strategie vir die produksie van geneesmiddels van S. runcinata.

O gênero Salvia L. (Lamiaceae), constituido de aproximadamente 900 espécies, possui 60 representantes no Brasil. Dentre suas propriedades medicinais destacam-se as atividades antitumoral, anti-inflamatória, cicatrizante e antibacteriana. Óleos essenciais e diterpenos de núcleos abietano, clerodano, primarano estão entre os principais constituintes químicos relatados para as espécies. O objetivo deste trabalho foi a caracterização química dos óleos essenciais obtidos por hidrodestilação de seis espécies de Salvia nativas da região Sul do Brasil (S. guaranitica, da Seção Coerulae; S. ovalifolia, S. cordata e S. borjensis, da Seção Rudes e S. procurrens e S. uliginosa, da Seção Uliginosae), utilizando cromatografia à gás/espectrometria de massas. A análise química dos óleos essenciais mostrou que todas as espécies apresentaram apenas sesquiterpenos, além de compostos alifáticos como aldeidos e ácidos graxos de cadeia longa. Entre estes últimos, nonadecanal estava presente em todas as espécies, sendo abundante (13% a 56%) em S. procurrens, S. ovalifolia e S. borjensis. Em relação à composição dos sesquiterpenos, observou-se que espatulenol estava presente em quantidades relativamente grandes (10% - 40%) nas espécies S. cordata, S. borjensis e S. ovalifolia. Estas espécies também apresentaram β-cariofileno, sesquiterpeno encontrado, ainda, em S. guaranitica. S. cordata apresentou o sesquiterpeno epi-globulol (32,3%) e 2E, 6Z-farnesol (11,4%). O óleo essencial de S. uliginosa apresentou ácido hexadecanóico (30,1%), n-nonadecano (17,5%) e nonadecanal (4,5%) como componentes majoritários. Os óleos essenciais e os extratos diclorometano das espécies da Seção Uliginosae (S. uliginosa e S. procurrens) mostraram atividade contra Staphylococcus aureus, quando testados pelo método de bioautografia. As analises preliminares sugerem a presença de diterpenos de núcleo abietano-quinona nos extratos orgânicos destas espécies. Os extratos diclorometano dessas duas espécies foram também avaliados quanto à atividade amebicida contra amebas de vida livre Acanthamoeba polyphaga. Entre estes, apenas o de S. uliginosa foi ativo, apresentando 100% de atividade nas concentrações de 7,5 e 5 mg/mL. / The genus Salvia L. (Lamiaceae) has about 900 species, 60 of them found in Brazil. Among the medicinal properties it is possible to highlight anti-tumor, anti-inflammatory, antioxidant and antibacterial activities. Essential oils and diterpenes with abietane, clerodane and primarane skeletons are the main componentes of these plants. In this sense, the purpose of this study was to characterize the essential oils obtained by hydrodistillation from Salvia species native to southern Brazil (S. guaranitica, Section Coerula; S. ovalifolia, S. cordata and S. borjensis, Section Rudes and S. procurrens and S. uliginosa, Section Uliginosae), using gas chromatography coupled to mass spectrometry. Chemical analysis of the essential oils showed that all species had only sesquiterpenes, and aliphatic compounds such as aldehydes and long chain fatty acids. Among the latter, nonadecanal was present in all species, being abundant (13% to 56%) in S. procurrens, S. borjensis and S. ovalifolia. Regarding the composition of the sesquiterpenes, it was observed that spathulenol was present in relatively large amounts (10% - 40%) in S. cordata, S. borjensis and S. ovalifolia oils. These species also showed β-caryophyllene, a sesquiterpene also found in S. guaranitica. S. cordata showed the sesquiterpene epi-globulol (32.3%) and 2E, 6Z-farnesol (11.4%). S. uliginosa essential oil presented hexadecanoic acid (30.1%), n-nonadecane (17.5%) and nonadecanal (4.5%) as the main compound. The essential oils and the dichloromethane extracts of of the species from the Section Uliginosae (S. uliginosa and S. procurrens) demonstrated antimicrobial activity against Staphylococcus aureus when investigated by the bioautography method. Preliminary analyses indicated theat the main compounds present in the organic extracts are diterpenes with the abietane type-diterpene quinone nucleous. The dichloromethane extracts of these two species were also evaluated for the activity against Acanthamoeba polyphaga. Only the extract of S. uliginosa was active, with 100% activity at concentrations of 7.5 and 5 mg/mL.

O gênero Salvia L. (Lamiaceae), constituido de aproximadamente 900 espécies, possui 60 representantes no Brasil. Dentre suas propriedades medicinais destacam-se as atividades antitumoral, anti-inflamatória, cicatrizante e antibacteriana. Óleos essenciais e diterpenos de núcleos abietano, clerodano, primarano estão entre os principais constituintes químicos relatados para as espécies. O objetivo deste trabalho foi a caracterização química dos óleos essenciais obtidos por hidrodestilação de seis espécies de Salvia nativas da região Sul do Brasil (S. guaranitica, da Seção Coerulae; S. ovalifolia, S. cordata e S. borjensis, da Seção Rudes e S. procurrens e S. uliginosa, da Seção Uliginosae), utilizando cromatografia à gás/espectrometria de massas. A análise química dos óleos essenciais mostrou que todas as espécies apresentaram apenas sesquiterpenos, além de compostos alifáticos como aldeidos e ácidos graxos de cadeia longa. Entre estes últimos, nonadecanal estava presente em todas as espécies, sendo abundante (13% a 56%) em S. procurrens, S. ovalifolia e S. borjensis. Em relação à composição dos sesquiterpenos, observou-se que espatulenol estava presente em quantidades relativamente grandes (10% - 40%) nas espécies S. cordata, S. borjensis e S. ovalifolia. Estas espécies também apresentaram β-cariofileno, sesquiterpeno encontrado, ainda, em S. guaranitica. S. cordata apresentou o sesquiterpeno epi-globulol (32,3%) e 2E, 6Z-farnesol (11,4%). O óleo essencial de S. uliginosa apresentou ácido hexadecanóico (30,1%), n-nonadecano (17,5%) e nonadecanal (4,5%) como componentes majoritários. Os óleos essenciais e os extratos diclorometano das espécies da Seção Uliginosae (S. uliginosa e S. procurrens) mostraram atividade contra Staphylococcus aureus, quando testados pelo método de bioautografia. As analises preliminares sugerem a presença de diterpenos de núcleo abietano-quinona nos extratos orgânicos destas espécies. Os extratos diclorometano dessas duas espécies foram também avaliados quanto à atividade amebicida contra amebas de vida livre Acanthamoeba polyphaga. Entre estes, apenas o de S. uliginosa foi ativo, apresentando 100% de atividade nas concentrações de 7,5 e 5 mg/mL. / The genus Salvia L. (Lamiaceae) has about 900 species, 60 of them found in Brazil. Among the medicinal properties it is possible to highlight anti-tumor, anti-inflammatory, antioxidant and antibacterial activities. Essential oils and diterpenes with abietane, clerodane and primarane skeletons are the main componentes of these plants. In this sense, the purpose of this study was to characterize the essential oils obtained by hydrodistillation from Salvia species native to southern Brazil (S. guaranitica, Section Coerula; S. ovalifolia, S. cordata and S. borjensis, Section Rudes and S. procurrens and S. uliginosa, Section Uliginosae), using gas chromatography coupled to mass spectrometry. Chemical analysis of the essential oils showed that all species had only sesquiterpenes, and aliphatic compounds such as aldehydes and long chain fatty acids. Among the latter, nonadecanal was present in all species, being abundant (13% to 56%) in S. procurrens, S. borjensis and S. ovalifolia. Regarding the composition of the sesquiterpenes, it was observed that spathulenol was present in relatively large amounts (10% - 40%) in S. cordata, S. borjensis and S. ovalifolia oils. These species also showed β-caryophyllene, a sesquiterpene also found in S. guaranitica. S. cordata showed the sesquiterpene epi-globulol (32.3%) and 2E, 6Z-farnesol (11.4%). S. uliginosa essential oil presented hexadecanoic acid (30.1%), n-nonadecane (17.5%) and nonadecanal (4.5%) as the main compound. The essential oils and the dichloromethane extracts of of the species from the Section Uliginosae (S. uliginosa and S. procurrens) demonstrated antimicrobial activity against Staphylococcus aureus when investigated by the bioautography method. Preliminary analyses indicated theat the main compounds present in the organic extracts are diterpenes with the abietane type-diterpene quinone nucleous. The dichloromethane extracts of these two species were also evaluated for the activity against Acanthamoeba polyphaga. Only the extract of S. uliginosa was active, with 100% activity at concentrations of 7.5 and 5 mg/mL.

O gênero Salvia L. (Lamiaceae), constituido de aproximadamente 900 espécies, possui 60 representantes no Brasil. Dentre suas propriedades medicinais destacam-se as atividades antitumoral, anti-inflamatória, cicatrizante e antibacteriana. Óleos essenciais e diterpenos de núcleos abietano, clerodano, primarano estão entre os principais constituintes químicos relatados para as espécies. O objetivo deste trabalho foi a caracterização química dos óleos essenciais obtidos por hidrodestilação de seis espécies de Salvia nativas da região Sul do Brasil (S. guaranitica, da Seção Coerulae; S. ovalifolia, S. cordata e S. borjensis, da Seção Rudes e S. procurrens e S. uliginosa, da Seção Uliginosae), utilizando cromatografia à gás/espectrometria de massas. A análise química dos óleos essenciais mostrou que todas as espécies apresentaram apenas sesquiterpenos, além de compostos alifáticos como aldeidos e ácidos graxos de cadeia longa. Entre estes últimos, nonadecanal estava presente em todas as espécies, sendo abundante (13% a 56%) em S. procurrens, S. ovalifolia e S. borjensis. Em relação à composição dos sesquiterpenos, observou-se que espatulenol estava presente em quantidades relativamente grandes (10% - 40%) nas espécies S. cordata, S. borjensis e S. ovalifolia. Estas espécies também apresentaram β-cariofileno, sesquiterpeno encontrado, ainda, em S. guaranitica. S. cordata apresentou o sesquiterpeno epi-globulol (32,3%) e 2E, 6Z-farnesol (11,4%). O óleo essencial de S. uliginosa apresentou ácido hexadecanóico (30,1%), n-nonadecano (17,5%) e nonadecanal (4,5%) como componentes majoritários. Os óleos essenciais e os extratos diclorometano das espécies da Seção Uliginosae (S. uliginosa e S. procurrens) mostraram atividade contra Staphylococcus aureus, quando testados pelo método de bioautografia. As analises preliminares sugerem a presença de diterpenos de núcleo abietano-quinona nos extratos orgânicos destas espécies. Os extratos diclorometano dessas duas espécies foram também avaliados quanto à atividade amebicida contra amebas de vida livre Acanthamoeba polyphaga. Entre estes, apenas o de S. uliginosa foi ativo, apresentando 100% de atividade nas concentrações de 7,5 e 5 mg/mL. / The genus Salvia L. (Lamiaceae) has about 900 species, 60 of them found in Brazil. Among the medicinal properties it is possible to highlight anti-tumor, anti-inflammatory, antioxidant and antibacterial activities. Essential oils and diterpenes with abietane, clerodane and primarane skeletons are the main componentes of these plants. In this sense, the purpose of this study was to characterize the essential oils obtained by hydrodistillation from Salvia species native to southern Brazil (S. guaranitica, Section Coerula; S. ovalifolia, S. cordata and S. borjensis, Section Rudes and S. procurrens and S. uliginosa, Section Uliginosae), using gas chromatography coupled to mass spectrometry. Chemical analysis of the essential oils showed that all species had only sesquiterpenes, and aliphatic compounds such as aldehydes and long chain fatty acids. Among the latter, nonadecanal was present in all species, being abundant (13% to 56%) in S. procurrens, S. borjensis and S. ovalifolia. Regarding the composition of the sesquiterpenes, it was observed that spathulenol was present in relatively large amounts (10% - 40%) in S. cordata, S. borjensis and S. ovalifolia oils. These species also showed β-caryophyllene, a sesquiterpene also found in S. guaranitica. S. cordata showed the sesquiterpene epi-globulol (32.3%) and 2E, 6Z-farnesol (11.4%). S. uliginosa essential oil presented hexadecanoic acid (30.1%), n-nonadecane (17.5%) and nonadecanal (4.5%) as the main compound. The essential oils and the dichloromethane extracts of of the species from the Section Uliginosae (S. uliginosa and S. procurrens) demonstrated antimicrobial activity against Staphylococcus aureus when investigated by the bioautography method. Preliminary analyses indicated theat the main compounds present in the organic extracts are diterpenes with the abietane type-diterpene quinone nucleous. The dichloromethane extracts of these two species were also evaluated for the activity against Acanthamoeba polyphaga. Only the extract of S. uliginosa was active, with 100% activity at concentrations of 7.5 and 5 mg/mL.

>Magister Scientiae - MSc / Salvia hispanica L. is an annual pseudocereal food crop, locally known as chia that has the ability to grow in water stress environments. The importance of chia dates back to the pre-columbian era where it was consumed as staple food by the indigenous South Americans due to its high nutritional and medicinal benefits. A single chia plant produces two seed variants: white seed genotype (denoted as WSG) and black seed genotype (denoted as BSG). Chia seeds have been proven to have a huge potential as a healthy food source and contained various medicinal properties. However, these plants are still prone to environmental stress conditions such as salinity that is one of the major abiotic stresses that influence crop production and yield worldwide. Despite the nutritional impact of the chia seeds, limited information regarding their molecular responses to abiotic stress conditions are known. This study was divided into two distinct parts. Firstly, the study comparatively analysed the leaf proteomes of two chia genotypes using gelbased proteomic analysis coupled with mass spectrometry. Total soluble proteins were extracted from chia leaves and subjected to 2-D PAGE analysis. Proteins were visualized by CBB and identified by MALDI-TOF MS/MS. A total of 284 and 209 spots were detected in WSG and BSG, respectively. Using mass spectrometry, 36 differentially expressed protein spots were successfully identified based on their protein abundance using homology database searches. Interestingly, two defensive-related proteins (osmotin-like protein and the chalcone isomerase) were only present in WSG and absent in BSG. In light of previous information regarding the nutritional profiles (no significant difference) of these two genotypes, this study has shown that there are distinct molecular differences between these genotypes. Therefore, WSG will be used in further downstream analysis. The second part of this study focused on the influence of salt stress (imposed by 100 mM NaCl) on the leaf proteome of WSG. Using gel-based proteomic analysis, 61 differentially expressed proteins were identified and classified into nine functional categories. Most of the proteins identified in this study were upregulated by salt stress. Interesting to note, 12 proteins identified in this study were only present in response to salt stress but were absent in the control. These proteins include ATP-dependent zinc metalloprotease FTSH 2 (spot 48), HSP70 proteins (spots 46 and 47), superoxide dismutases (spots 10, 41 and 42) and an ascorbate peroxidase (spot 56). All these proteins are important antioxidants that play a significant role in scavenging reactive oxygen species (ROS). Previous studies have shown that these antioxidants play vital roles in stress tolerance. These proteins could serve as potential biomarkers that could be used to enhance salt stress tolerance in pseudocereals and cereal food crops. / National Research Foundation (NRF) and Agricultural Research Council

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-12-21T11:16:37Z No. of bitstreams: 1 texto completo.pdf: 1681764 bytes, checksum: a709e0d8e716b792d8e43a17aae07bac (MD5) / Made available in DSpace on 2016-12-21T11:16:37Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1681764 bytes, checksum: a709e0d8e716b792d8e43a17aae07bac (MD5) Previous issue date: 2016-02-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A chia (Salvia hispanica L.) vem sendo consumida pela população mundial devido às suas propriedades funcionais e alta concentração de ácidos graxos essenciais, fibras e proteínas. No entanto, é necessário caracterizar a concentração de nutrientes nas sementes de chia cultivadas no Brasil. Assim, o presente estudo objetivou analisar a ocorrência e concentração de macro e micronutrientes, carotenoides, flavonoides, ácido fítico e tanino em sementes de chia cultivadas em dois locais distintos, assim como avaliar a qualidade proteica e a biodisponibilidade de ferro nas sementes, uma vez que estudos nesse linha de pesquisa são escassos. Foram utilizadas sementes de chia cultivados em Catuípe, Rio Grande do Sul (RS) e Jaciara, Mato Grosso (MT). As sementes foram moídas e armazenadas em sacos de polietileno recobertos com papel alumínio em freezer até o momento das análises. Foi determinada a concentração de umidade, lipídios, proteínas, carboidratos, fibra alimentar total, cinzas totais e minerais presentes nas sementes de chia. A investigação de carotenoides (luteína e zeaxantina), vitamina C (ácido ascórbico), flavonoides (3-deoxiantocianidinas - 3- DXAs, flavonas e flavanonas) foi realizada por cromatografia líquida de alta eficiência (CLAE), com detecção por arranjo de diodos, enquanto a análise de vitamina E (α, , , δ tocoferóis e tocotrienois) foi feita por CLAE, com detecção por fluorescência. A capacidade antioxidante, concentração de fenólicos totais, fitato e taninos foram realizados por espectrofotometria. Além disso, foi avaliada em ratos Wistar, a qualidade proteica da semente e farinha de chia por meio do coeficiente de eficiência alimentar (CEA), coeficiente de eficiência proteica (PER), razão proteica líquida (NPR) e digestibilidade verdadeira (DV) e a biodisponibilidade de ferro da farinha, utilizando o método de depleção/repleção de hemoglobina. Para análise estatística dos compostos presentes nas sementes na chia, foi utilizado o teste t para comparação de duas amostras. Para a análise da qualidade proteica, os dados obtidos foram submetidos à ANOVA. As médias dos grupos testes foram comparadas pelo teste de Duncan. No ensaio de biodisponibilidade de ferro, para avaliar se houve diferença entre os grupos experimentais quanto ao ganho de peso, CEA, ganho de hemoglobina e marcadores moleculares, foram realizados a ANOVA e o teste de Newman Keulls. O nível de significância estabelecido para todos os testes foi 5%. Os dados foram analisados com auxílio do software estatístico SPSS, versão 20.0. A chia apresentou alta concentração de lipídios (31,2 g.100 g -1 , em média), proteínas (18,9 g.100 g -1 , em média) e fibra alimentar total (35,3 g.100 g -1 , em média). A chia cultivada no RS apresentou maior (p≤0,05) concentração de ferro, manganês, boro, chumbo, alumínio, nitrogênio e fósforo. A concentração de vitamina E total nas sementes de chia foi alta (7038,43 μg.100 g -1 e 7024,59 μg.100 g -1 para a sementes cultivada no RS e MT, respectivamente), sendo -tocoferol o componente principal encontrado (7031,51 ± 1β9,54 μg.100 g -1 , em média). Os carotenoides foram identificados apenas na semente cultivada no RS e a ocorrência de vitamina C e 3- DXAs não foi observada nas duas sementes. Observou-se valores semelhantes de compostos fenólicos totais e ácido fítico nas sementes de chia (p>0,05). A concentração de taninos foi superior (p≤0,05) na chia cultivada no Mato Grosso (19,08 ± 1,08 eq. catequina/g amostra) em relação à cultivada no Rio Grande do Sul (14,93 ± 0,24 eq. catequina/g amostra). A chia cultivada no RS apresentou a maior (p≤0,05) atividade antioxidante (478,2 ± 0,02 μ mol TEAC/g de amostra) em relação à cultivada no MT. Os valores de PER, NPR e DV não diferiram (p>0,05) entre os animais alimentados com diferentes formas de tratamento da chia e foram inferiores ao grupo controle (caseína) (p≤0,05). Os animais que foram alimentados com dietas testes apresentaram concentrações de glicose, triacilglicerídeos (TGL) e lipoproteína de muito baixa densidade (VLDL) inferiores (p≤0,05) e lipoproteína de alta densidade HDL superior (p≤0,05) aos animais que consumiram caseína. O peso do fígado dos animais alimentados com chia não diferiu (p>0,05) e foi inferior (p≤0,05) ao grupo controle. A espessura das camadas musculares do intestino e a profundidade das criptas foram superiores (p≤0,05) nos grupos alimentados com chia. No experimento biodisponibilidade do ferro observou-se consumo de ferro e ingestão total menores nos animais que receberam dieta rica em gordura (p≤0,05). O ganho de peso corporal, concentração de hemoglobina, aumento de hemoglobina, eficiência de regeneração da hemoglobina (HRE%) e valor biológico da eficiência de regeneração da hemoglobina (RBV-HRE) não diferiu (p>0,05) entre os grupos experimentais (p>0,05). O grupo alimentado com dieta padrão + chia (SD + C) apresentou menor expressão de transferrina, quando comparado com o grupo de controle (dieta padrão + sulfato ferroso) (p≤0,05). A expressão da ferritina foi inferior (p≤0,05) em todos os grupos experimentais quando comparados ao controle. A expressão do gene peroxissoma proliferador ativado por receptor α (PPAR-α) em animais alimentados com SD + C foi maior do que no grupo de controle (p≤0,05). A expressão gênica de citocromo b duodenal (DcytB) e transportador de metal divalente (DMT-1) foi maior (p ≤ 0,05) no grupo alimentado com dieta hiperlipídica + chia (HFD + C). No entanto, a expressão de hephaestina foi inferior (p≤0,05) em todos os grupos experimentais em comparação com o grupo controle e a expressão gênica de ferroportina foi inferior (p≤0,05) nos animais alimentados com chia. Animais alimentados com a dieta rica em gordura apresentaram biodisponibilidade do ferro da chia semelhante aos animais alimentados com uma dieta padrão. Em conclusão, a chia apresentou elevada concentração de vitamina E, ácidos graxos poli-insaturados, fibra alimentar, atividade antioxidante, ferro, cálcio, manganês e zinco. Além disso, o consumo de chia por ratos resultou em boa digestibilidade proteica, efeito hipoglicêmico e melhora no perfil lipídico, reduzindo, ainda, a deposição de gordura no fígado dos animais em um curto período de tempo (28 dias), e também promoveu alterações no tecido intestinal, aumentando a sua funcionalidade. Animais alimentados com dieta rica em gordura apresentaram biodisponibilidade do ferro da chia semelhante aos animais alimentados com dieta padrão. / Chia (Salvia hispanica L.) has been consumed by the world population due to their functional properties and high concentration of essential fatty acids, dietary fiber and protein. However, it is necessary to characterize the nutrient concentration in chia seeds grown in Brazil. The present study aimed to analyze the occurrence and concentration of macro and micronutrients, carotenoids, flavonoids, phytic acid and tannin in chia seeds grown in two different places, as well as evaluating the protein quality and bioavailability of iron in the seeds, since studies in this research field are scarce. Chia seeds used were grown in Catuípe, Rio Grande do Sul (RS) and Jaciara, Mato Grosso (MT). The seeds were ground and stored in polyethylene bags covered with foil in a freezer until the moment of analysis. The concentration of moisture, lipids, proteins, carbohydrates, dietary fiber, minerals and total ash present in chia seeds was determined. The carotenoid investigation (lutein and zeaxanthin), vitamin C (ascorbic acid), flavonoids (3-deoxiantocianidins - 3-DXAs, flavones, flavanones) was performed by high-performance liquid chromatography (HPLC) with detection by diode array, while vitamin E analysis (α, , , δ tocopherols and tocotrienol) was made by HPLC with fluorescence detection. The antioxidant capacity, concentration of total phenolics, phytate and tannins were carried out by spectrophotometry. Furthermore, was available in Wistar mice the protein quality of chia seed and chia flour by feed efficiency ratio (FER), protein efficiency ratio (PER), net protein ratio (NPR) and true digestibility (TD) and iron bioavailability was evaluated using the hemoglobin depletion/ repletion method in animals fed with standard diet and high fat diet. For the analysis of compounds present in chia seeds, we used the t test to compare two samples. For statistical analysis of protein quality, data were submitted to ANOVA. The average test groups were compared by Duncan test. The iron bioavailability experiment, to evaluate the differences between the groups on weight gain, CEA, hemoglobin gain and molecular analysis it were performed ANOVA and the Newman Keulls tests. The significance level for all tests was 5%. Data were analyzed using the statistical software SPSS, version 20.0. There were high lipid (31.2 g.100 g -1 , on average), proteins (18.9 g.100 g -1 , on average) and total dietary fiber (35.3 g.100 g -1 , on average) concentrations in chia seeds. The chia that has grown in RS had a higher (p<0.05) concentration of iron, manganese, boron, lead, aluminum, nitrogen and phosphorus. The concentration of total vitamin E in the chia seeds was high (70γ8.4γ μg.100 g -1 and 7024.59 μg.100 g -1 for the seed grown in RS and MT, respectively), being -tocopherol the major component found (7031.51 ± 1β9.54 μg.100 g -1 , on average). The carotenoids were identified only in the chia grown in RS and the occurrence of vitamin C and 3-DXAS was not observed in the chia seed grown in RS and MT. Similar values of total phenolic compounds and phytic acid were observed in the chia seeds (p>0.05). The concentration of tannins was higher (p<0.05) in the seed grown in Mato Grosso (19.08 ±1.08 eq. catequina/g sample) than in the seed grown in Rio Grande do Sul (14.93 ± 0.24 eq. catequina/g sample). Chia grown in RS showed the highest (p<0.05) antioxidant activity (478.2 ± 0.02 μmol TEAC/g sample) compared to that grown in MT. In the study of protein quality it were used chia seeds and chia flour with and without heat treatment. The values of PER, NPR and DV did not differed (p>0.05) among the animals that were fed with chia and were lower than the control group (casein). The animals fed with tests diets showed lower concentrations of glucose, triacylglycerides (TGL) and very low density lipoprotein (VLDL) and higher concentrations of high-density lipoprotein (HDL) (p<0.05) than the control group. The liver weights of animals that were fed with chia seed and chia flour did not differed (p>0.05) and were lower (p<0.05) than the control group. Crypt depth and thickness of intestinal muscle layers were higher (p<0.05) in groups that were fed with chia seed and chia flour. The iron bioavailability experiment of chia flour showed that the animals fed with high fat diet had similar iron bioavailability of chia compared to animals fed with standard diet. The total consumption and iron intake were lower in animals which received high fat diet (p<0.05). Body weight gain, hemoglobin concentrations, hemoglobin gain, hemoglobin regeneration efficiency (HRE%) and biological value of hemoglobin regeneration efficiency (RBV-HRE) did not differed among the experimental groups (p>0.05). The standard diet + chia (SD + C) group showed lower expression of transferrin when compared to the control group (standard diet + ferrous sulfate) (p<0.05). Ferritin expression was lower (p<0.05) in all experimental groups when compared to the control. The peroxisome proliferator-activated receptor-α (PPAR-α) gene expression in animals fed with SD + C was higher than in the control group (p≤0.05). The mRNA expression of duodenal cytochrome b (DcytB) and divalent metal transporter (DMT- 1) was higher (p<0.05) in the high fat diet + chia (HFD + C) group. However, hephaestin expression was lower (p<0.05) in all experimental groups compared to the control group and the gene expression of ferroportin was lower (p<0.05) in the groups fed with chia flour. In conclusion, chia exhibits high concentration of vitamin E, polyunsaturated fatty acids, dietary fiber, antioxidant activity, iron, calcium, manganese and zinc. Moreover, the consumption of chia showed good protein digestibility, hypoglycemic effect, and improved the lipid profile, reduced fat deposition in the liver of animals in a short period of time (28 days), and also promoted alterations in the intestinal tissue, which increased its functionality. Animals fed with high fat diet showed similar iron bioavailability of chia compared to animals fed with standard diet.

The thesis deals with particular green spice like Mentha longifolia L., Mentha spicata L., Mentha tomentosa Sm. 'Fíková', Mentha suaveolens Ehrt. 'Jablečná', Mentha piperita 'Persephone', Origanum vulgare L., Origanum heracleoticum L., Origanum vulgare L.'Aureum', Origanum vulgare L. 'Thumbles variety', Satureja montana L., Satureja hortensis L., Salvia glutinosa L., Salvia verbanaca L., Salvia verticilata L., Salvia lyrata L., Salvia przewalskii Maxim, Salvia officinalis L. 'Purpurascens', Allium nutans L., Allium stipitatum L., Allium tuberosum Rottl. Ex Spreng. In theoretic part a botanical charakteristics of the spice, legislation concerning the green spice, new information about vitamin C and factors influencing the content of ascorbic acid in plants are described. In practical part, the content of vitamin C in vegetative and generative growth phase has been determined. Results have been evalueted and compared.

En esta tesis se estudió el fraccionamiento de aceites esenciales obtenidos de plantas aromáticas comunes en el país mediante el uso de dióxido de carbono supercrítico, con el objetivo de obtener compuestos o fracciones con actividad biocida. Se seleccionaron tres plantas: Tagetes minuta, Salvia officinalis y Mentha piperita. La extracción del aceite esencial se realizó mediante hidrodestilación, determinándose su composición mediante cromatografía de gases. Como parte de esta tesis, se construyó un equipo experimental de alta presión, con el cual se realizaron mediciones de solubilidad y composición de la fase supercrítica para cada sistema [CO2 + aceite esencial] en distintas condiciones de presión y temperatura (70–120 bar, 313–323 K), utilizando el método dinámico o de gas saturado. Se realizaron también ensayos de fraccionamiento semicontinuo para determinar la influencia de la composición en el grado de separación. Con los resultados experimentales obtenidos se validó un modelo termodinámico predictivo basado en la ecuación de estado a contribución grupal (GC-EOS), previo ajuste de los parámetros necesarios con datos provenientes de otras fuentes. Utilizando dicho modelo como base, y reduciendo la complejidad de los sistemas en términos de dos o tres pseudo-componentes, se realizaron simulaciones de columnas de fraccionamiento de múltiples etapas en contra-corriente, analizando la influencia de las variables operativas (presión, temperatura, relación solvente/ alimentación, relación de reflujo, etc.) con el objetivo de hallar condiciones recomendables de operación para separar los compuestos biocidas de interés. En el análisis, se utilizaron herramientas y consideraciones de la Ingeniería del Equilibrio de Fases. En el caso del aceite de T. minuta se procuró separar la fracción de cetonas insaturadas (ocimenonas); en el caso del aceite de S. officinalis se planteó la purificación de la fracción de monoterpenos oxigenados, separándolos a la vez de los hidrocarburos y de los sesquiterpenos; en el caso del aceite de M. piperita se planteó la separación de mentona y mentol. La actividad biocida de las fracciones obtenidas experimentalmente a partir del aceite de T. minuta fueron ensayadas frente a dos parásitos: el gorgojo del maíz y el nemátodo del nudo de la raíz. / The fractionation of essential oils from local aromatic species using supercritical carbon dioxide was studied, in order to obtain compounds or fractions with biocidal activity. Three species were chosen: Tagetes minuta, Salvia officinalis y Mentha piperita. The essential oil was extracted by hydrodistillation and its composition determined by gas chromatography. As a part of this project, a lab-scale high-pressure experimental apparatus was built. With this equipment, measurements of solubility and composition in the supercritical phase were performed for each [CO2 + essential oil] system at different pressure and temperature conditions (70–120 bar, 313–323 K), using a dynamic or gas saturated method. Semi-continuous fractionation experiments were also carried out in order to determine the effect of composition in the separation performance. With the experimental data a predictive thermodynamic model based on the group contribution equation of state (GC-EOS) was validated, after fitting the necessary parameters with data from other sources. With this model as background, and reducing the systems complexity in terms of two or three pseudo-components, simulations of multistage counter-current columns were carried out, analyzing the influence of the operation parameters (pressure, temperature, solvent-to-feed ratio, reflux ratio, etc.), in order to find recommended conditions for the separation of the biocidal components. In this analysis, phase equilibrium engineering tools and considerations were applied. In the case of T. minuta, the goal was to isolate the unsaturated ketones fraction (ocimenones); in the case of S. officinalis the goal was the separation of the oxygenated monoterpenes fraction from both the hydrocarbon and the sesquiterpene fraction; finally in the case of M. piperita the separation between menthone and menthol was proposed. The biocidal activity of the fractions obtained experimentally from T. minuta oil was tested against the corn weevil and the root knot nemathod.

Tam Wing Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 82-100). / Abstracts in English and Chinese. / English abstrac --- p.I / 中文摘要 --- p.VI / Glossary --- p.X / Chapter Chapter 1. --- Background: / Chapter 1.1. --- Coronary heart disease in Hong Kong --- p.1 / Chapter 1.2. --- Coronary heart disease and atherosclerosis --- p.2 / Chapter 1.3. --- Pathogenesis of atherosclerosis --- p.2 / Chapter 1.4. --- Risk factors for atherosclerosis --- p.5 / Chapter 1.5. --- Homocysteine --- p.6 / Chapter 1.6. --- Folate --- p.10 / Chapter 1.7. --- Vitamin B12 --- p.13 / Chapter 1.8. --- Adhesion Molecules --- p.14 / Chapter 1.9. --- Phytoestrogen --- p.17 / Chapter 1.10. --- Secondary prevention of coronary artery disease --- p.20 / Chapter Chapter 2. --- "Heart disease, Danshen and Gegen in Chinese medicine" / Chapter 2.1. --- The record of Cardiac symptoms in Chinese Medicine --- p.24 / Chapter 2.2. --- Danshen (Salvia Miltriorrhiza) --- p.25 / Chapter 2.3. --- Gegen (Radix Pueraria) --- p.28 / Chapter Chapter 3. --- Surrogate atherosclerotic markers / Chapter 3.1. --- Flow-mediated dilatation of brachial artery (FMD) --- p.31 / Chapter 3.2. --- Carotid intima media thickness (IMT) --- p.32 / Chapter Chapter 4. --- Method / Chapter 4.1. --- Rational of the study --- p.33 / Chapter 4.2. --- Clinical protocol --- p.35 / Chapter 4.3. --- Measurement of plasma homocysteine --- p.38 / Chapter 4.4. --- Measurement of folate and vitamin B12 --- p.40 / Chapter 4.5. --- Measurement of soluble cellular adhesion molecules (CAMs) --- p.41 / Chapter 4.6. --- Measurement of plasma enterolactone --- p.43 / Chapter 4.7. --- Measurement of plasma hs-C-reactive protein --- p.44 / Chapter 4.8. --- Other laboratory tests --- p.45 / Chapter 4.9. --- High resolution ultrasound imaging --- p.46 / Chapter 4.10. --- Statistical analysis --- p.49 / Chapter 4.11. --- My contribution to this joint project --- p.49 / Chapter Chapter 5. --- Results / Chapter 5.1. --- Recruitment and outcomes of subjects --- p.51 / Chapter 5.2. --- Baseline characteristics --- p.53 / Chapter 5.3. --- Medical history and treatment received in the study subjects --- p.54 / Chapter 5.4. --- Safety profiles --- p.55 / Chapter 5.5. --- Severe adverse events --- p.56 / Chapter 5.6. --- Lipid profiles --- p.57 / Chapter 5.7. --- Secondary endpoints --- p.58 / Chapter 5.8. --- Primary endopoints --- p.59 / Chapter 5.9. --- The effect of statin usage on the primary endpoints / Chapter 5.10. --- The major determinant of the change in FMD by multivariate logistic regression / Chapter 5.11. --- Progress of lipid profiles and primary endpoints in the open label phase / Chapter Chapter 6. --- Discussion / Chapter 6.1. --- Brachial FMD --- p.66 / Chapter 6.2. --- Carotid IMT --- p.69 / Chapter 6.3. --- Brachial GTN --- p.70 / Chapter 6.4. --- Lipid-lowering effect --- p.72 / Chapter 6.5. --- Phytoestrogen --- p.72 / Chapter 6.6. --- Folate --- p.73 / Chapter 6.7. --- Vitamin B12 and glucose --- p.76 / Chapter 6.8. --- Summary of possible anti-atherogenic mechanism of D&G --- p.76 / Chapter 6.9. --- Placebo effect --- p.77 / Chapter 6.10. --- Safety profile --- p.77 / Chapter 6.11. --- Limitation of the study and suggestion of solution --- p.77 / Chapter 6.12. --- Suggestions and ummary of the future work --- p.79 / Chapter Chapter 7. --- Conclusions --- p.81 / References --- p.82