Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains x, 123 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 113-123).

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / This study: 1. Describes the preparation of unfermented and fermented rooibos methanol and aqueous extracts. 2. Investigates the influence of unfermented and fermented rooibos methanol and aqueous extracts on the binding of natural steroid substrates to ovine adrenal microsomal cytochrome P450 enzymes, demonstrating that the binding of natural steroids is inhibited in the presence of rooibos extracts. 3. Describes an assay demonstrating the inhibitory effect of rooibos extracts on the catalytic activity of cytochrome 17α-hydroxylase (CYP17) and cytochrome 21-hydroxylase (CYP21) in ovine adrenal microsomes. 4. Investigates the influence of unfermented and fermented rooibos methanol extracts on the catalytic activity of individual cytochrome P450 enzymes – CYP17 and baboon CYP21, that are expressed in COS1 cells. 5. Demonstrates that fractions of the unfermented rooibos methanol extract inhibits the binding of natural steroid substrate to microsomal cytochrome P450 enzymes as well as the catalytic activity of baboon CYP21 expressed in COS1 cells. 6. Investigates the inhibitory influence of individual rooibos flavonoids on the catalytic activity of baboon CYP21 expressed in COS1 cells.

Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study describes: I. The cloning of the CVP 19 gene and construction of the intracellular expression vector pPIC3.5K-CYP19. II. The transformation of the yeast, Pichia pastoris with the constructed vector. III. The expression ofP450arom in Pichia pastoris. IV. The determination of enzyme activity and isolation of the protein from the Pichia pastoris cells. V. The expression of P450c 17 in Pichia pastoris. VI. The determination of kinetic constants for the conversion of progesterone to 170H-progesterone and 160H-progesterone by P450c17. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: I. Die klonering van die CVP 19 geen en die konstruksie van die intrasellulêre uitdrukkingsplasmied, pPIC3.5K-CYPI9. II. Die transformasie van die gis, Pichia pastoris, met die gekonstrueerde plasmied. III. Die uitdrukking van aromatase in Pichia pastoris. IV. Die bepaling van ensiemaktiwiteit en die isolering van die proteïen vanuit Pichia pastoris. V. Die uitdrukking van P450c17 in Pichia pastoris. VI. Die bepaling van kinetiese konstantes vir die omsetting van progesteroon na 170H-progesteroon en 160H-progesteroon deur P450c17.

Thesis (MSc) Stellenbosch University, 2014 / ENGLISH ABSTRACT: In this study, the effect of the amino acid residues at positions 40 and 407 on the catalytic activity of porcine CYP17A1 was investigated. Porcine cofactor CYB5 was cloned from porcine liver tissue and its effect on the catalytic activity of porcine CYP17A1 was determined. The influence of rat, human and angora CYB5 on the lyase activity of porcine CYP17A1 was subsequently determined and compared to the influence of porcine CYB5. Wt porcine CYP17A1, which has residues Val40 and His407, catalysed the conversion of prog efficiently with ~50% prog converted to 17OHprog (~40%) and A4 (~10%) after 3 hr. After 24 hr, negligible levels prog remained with ~71% 17OHprog and ~25% A4 being produced. Low levels of 16OHprog were formed (~9%). The Leu105Ala mutation reduced wt 17α-hydroxylase activity, with 70% prog remaining after 24 hr while 16OHprog (~10%) levels remained unchanged. Porcine CYP17A1 with residues Leu40 and His407, exhibited similar catalytic activity towards prog as did wt porcine CYP17A1 (Val40 and His407 residues), while porcine CYP17A1 with residues Leu40 and Leu407 increased the formation of A4 2-fold to 54% at 24 hr and porcine CYP17A1 with residues Val40 and Leu407 resulted in the highest formation of A4 (90%). Wt porcine CYP17A1, while having converted 95% of the prog substrate, produces only ~16% A4 after 24 hr. In the presence of porcine CYB5, however, the lyase activity was stimulated with 85% of prog being converted to A4 and only 13% 17OHprog remaining. The lyase activity was also stimulated by CYB5 from other species, resulting in an increase in A4 production of 60.6%, 24% and 11.6% by rat, angora and human CYB5, respectively. The degree of lyase stimulation correlated to the percentage identity of the CYB5 amino acid sequences to porcine CYB5. While the Val and Leu residues at position 40 do not appear to influence the lyase activity of porcine CYP17A1 as prominently as the residue at position 407, it is the charged residue at 407 that plays a significant role in the production of A4, decreasing A4 production irrespective of the Val and the Leu residues at position 40. It would, furthermore, appear that the stimulation of lyase activity of CYP17A1 is the greatest when assaying this activity in the presence of CYB5 of the same species as was detected when co-expressing porcine CYP17A1 and porcine CYB5. / AFRIKAANSE OPSOMMING: In hierdie studie is die invloed van die aminosuurresidue by posisies 40 en 407 op die katalitiese aktiwiteit van vark CYP17A1 ondersoek. Vark CYB5 is geklooneer vanuit vark lewer weefsel en die effek van hierdie kofaktor op die katalitiese aktiwiteit van vark CYP17A1 is bepaal. Die invloed van rot, mens en angora CYB5 op die liase aktiwiteit van vark CYP17A1 is daarna bepaal en vergelyk met die invloed van vark CYB5. Vark CYP17A1-VH, (kodeer Val40 en His407), kataliseer die omskakeling van prog doeltreffend met ~50 % prog wat omgeskakel word na 17OHprog (~40%) en A4 (~10%) na 3 uur. Na 24 uur, is feitlik alle prog omgeskakel, met ~71% 17OHprog en ~25% A4 geproduseer. Lae vlakke 16OHprog is ook gevorm (~9%). Die Leu105Ala mutasie verminder 17α- hidroksilase aktiwiteit, met 70% prog wat na 24 uur nie omgesit is nie, terwyl 16OHprog (~10%) vlakke onveranderd gebly het. Vark CYP17A1-LH (kodeer Leu40 en His407), en CYP17A1-VH het diselfde katalitiese aktiwiteit teenoor prog getoon, terwyl vark CYP17A1-LL (kodeer Leu40 en Leu407) die vorming van A4 2-voudig verhoog het tot 54% na 24 uur. Vark CYP17A1-VL (kodeer Val40 en Leu407) se katalitiese aktiwiteit het gelei tot die hoogste vorming van A4 (90%). Alhoewel CYP17A1-VH, 95% van die prog substraat omgeskakel het is slegs ~16% A4 geproduseer na 24 uur. In die teenwoordigheid van vark CYB5 is die liase aktiwiteit egter gestimuleer, en is 85% van die prog substraat omgeskakel na A4 met slegs 13% 17OHprog teenwoordig na 24 uur. Die liase aktiwiteit is ook gestimuleer deur CYB5 van ander spesies, wat lei tot 'n toename in A4 produksie van 60,6% , 24% en 11,6% deur rot, angora en menslike CYB5, onderskeidelik. Daar is gevind dat daar’n sterk korrelasie is tussen die stimulering van die liase aktiwitieit en die persentasie aminosuur volgorde identiteit van CYB5 afkomstig vanaf die verskillende spesies. Terwyl die Val en die Leu aminosuurresidu op posisie 40 wel die liase aktiwitiet tot ‘n mate beȉnvloed, blyk dit uit die data dat die potitief gelaaide residue by 407 'n belangrike rol speel in die produksie van A4, en A4 produksie verlaag ongeag van die Val en die Leu residu by posisie 40. Dit wil ook verdermeer voorkom asof die stimulering van die liase aktiwiteit van CYP17A1 die hoogste is wanneer die ensiem gekataliseerde reaksie deurgevoer word in die teenwoordigheid van CYB5 en CYP17A1 afkomstig vanaf dieselfde spesies.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: - the characterization and comparison of the enzymatic activity of both Angora and ovine 3βHSD expressed in non-steroidogenic COS-1 cells. The apparent Km and Vmax values for the metabolism of PREG, 17-OHPREG and DHEA were determined; - the characterization of steroid metabolites produced by COS-1 cells coexpressing either Angora or ovine 3βHSD together with Angora CYP17, in the presence and absence of overexpressed Cyt-b5, following the metabolism of PREG and 17-OHPREG. 3βHSD was identified as an additional factor in causing hypocortisolism in the South African Angora goat; - the influence of Cyt-b5 on the enzymatic activity of both Angora and ovine 3βHSD coexpressed in non-steroidogenic COS-1 cells; - the influence of purified ovine live Cyt-b5 and anti-Cyt-b5 IgG on adrenal microsomal 3βHSD activity. Cyt-b5 was shown to specifically augment 3βHSD activity which represents the first documentation of such augmentation in any species; - the overexpression and purification of Angora 3βHSD using a baculovirus expression system coupled with a detergent based enzyme purification method; - the characterization of both substrate and co-factor kinetics for the individual dehydrogenase and isomerase activities of purified 3βHSD, in the presence and absence of purified ovine liver Cyt-b5. Cyt-b5 was shown to increase the affinity of 3βHSD towards NAD+ during the dehydrogenase reaction whilst having no significant influence on the isomerase reaction. This represents the first documentation of Cyt-b5 influencing co-factor binding in any member of the -ydroxysteroid dehydrogenases; - the FRET analysis of COS-1 cells coexpressing 3βHSD-eCFP and Cyt-b5-eYFP fusion proteins, suggesting an allosteric interaction between 3βHSD and Cyt-b5. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: - die karakterisering en vergelyking van die ensiematiese aktiwiteit van beide Angora en skaap 3βHSD, wat uitgedruk was in nie-steroïed genererende COS-1 selle. Die Km en Vmax waardes tydens die metabolisme van PREG, 17-OHPREG en DHEA was bepaal; - die karakterisering van steroïed metaboliete gegenereer deur COS-1 selle wat Angora of skaap 3βHSD uitdruk saam met Angora CYP17, in die aanwesigheid of afwesigheid van sitochroom b5, na die metaboliseering van PREG en 17-OHPREG. 3βHSD was geïdentifiseer as ‘n bydraende faktor in die oorsaak van hipokortisolisme in die Suid-Afrikaanse Angorabok; - die invloed van sitochroom b5 op die ensiematiese aktiwiteit van beide Angora en skaap 3βHSD wat saam uitgedruk was in nie-steroïed genererende COS-1 selle; - die invloed van gesuiwerde skaap lewer sitochroom b5 en sitochroom b5 teenstof op mikrosomale 3βHSD aktiwiteit. Dit is getoon dat sitochroom b5 die aktiwiteit van 3βHSD spesifiek verhoog. Hierdie studie verteenwoordig die eerste dokumentasie van so ‘n verhoging in enige spesie; - die uitdrukking en suiwering van Angora 3βHSD deur middel van ‘n bakulo-virus sisteem gekoppel aan ‘n detergent gebaseerde ensiem suiwerings metode; - die karakterisering van beide substraat en ko-faktor kinetika vir die afsonderlike dehidrogenase en isomerase aktiwiteite van gesuiwerde 3βHSD, in die aanwesigheid of afwesigheid van gesuiwerde sitochroom b5. Dit is getoon dat sitochroom b5 die affiniteit van 3βHSD teenoor NAD+ tydens die dehidrogenase reaksie verhoog sonder om ‘n beduidende invloed op die isomerase reaksie te hê. Hierdie studie verteenwoordig die eerste dokumentasie van sitochroom b5 wat ko-faktor binding beïnvloed in enige lid van die hidroksisteroïed dehidrogenase familie van ensieme; - die analise van FRET sein in COS-1 selle wat beide 3βHSD-eCFP en Cyt-b5- eYFP fusie proteïene uitdruk. Die resultate stel voor dat sitochroom b5 3βHSD aktiwiteit beïnvloed deur middel van ‘n allosteriese meganisme.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: This study: 1. Describes the localization of CYP11B1 in the Cape baboon adrenal gland using Western blot analysis. CYP11B1 was localized to the adrenal cortex and medulla. 2. Describes the catalytic activity of CYP11B1 towards 11-deoxycorticosterone and corticosterone in adrenal cortical- and medullary tissue homogenates. Aldosterone formation in the adrenal medulla was identified using an atmospheric pressure chemical ionization-mass spectrometry method, which was developed in our department. 3. Compares the catalytic activity of three recombinant Cape baboon CYP11B1 cDNAs, expressed in COS-1 cells, towards 11-deoxycorticosterone and 11-deoxycortisol. 4. Describes the determination of the Michaelis-Menten constants and maximum reaction rates of 11-deoxycorticosterone and 11-deoxycortisol utilization by two functional recombinant Cape baboon CYP11B1 cDNAs, respectively. 11-Deoxycorticosterone metabolites were quantified using an enzyme immunoassay kit. 11-Deoxycortisol metabolites were quantified using a liquid chromatography-mass spectrometry method, which was developed in our department. 5. Describes the homology modeling of two isoforms of Cape baboon CYP11B1 using CYP102 and CYP2C5 as structural templates. The influence of three amino acid residue substitutions, located in the predicted D-E helix, on the catalytic activity of the two CYP11B1 isoforms was examined. / AFRIKAANSE OPSOMMING: Hierdie studie: 1. Beskryf die lokalisering van CYP11B1 in die bynier van die Kaapse bobbejaan deur gebruik te maak van die Western kladtegniek. CYP11B1 is gelokaliseer tot die adrenale korteks en medulla. 2. Beskryf die metabolisme van 11-deoksikortikosteroon en kortikosteroon in adrenale korteks- and medulla weefsel preparate, onderskeidelik. Die produksie van aldosteroon in die medulla is geïdentifiseer deur gebruik te maak van ‘n atmosferiese druk chemiese ionisasie-massa spektrometrie metode wat in ons departement ontwikkel is. 3. Vergelyk die katalitiese aktiwiteit van drie rekombinante Kaapse bobbejaan CYP11B1 cDNAs, getransfekteer in COS-1 selle, ten opsigte van 11-deoksikortikosteroon en 11- deoksikortisol metabolisme. 4. Beskryf die bepaling van die Michaelis-Menten konstantes en maksimum snelhede van twee funksionele rekombinante Kaapse bobbejaan CYP11B1 cDNAs, getransfekteer in COS-1 selle, ten opsigte van 11-deoksikortikosteroon en 11-deoksikortisol metabolisme. 11-Deoksikortikosteroon metaboliete is gekwantifiseer deur gebruik te maak van ‘n ensiem immunotoets. 11-Deoksikortisol metaboliete is gekwantifiseer deur middel van ‘n vloeistofchromatografie-massaspektrometrie metode, ontwikkel in ons departement. 5. Beskryf die modelering van drie-dimensionele strukture van twee funksionele Kaapse bobbejaan CYP11B1 isoensieme deur CYP102 en CYP2C5 as template te gebruik. Die effek van drie aminosuurresiduveranderinge in die voorspelde D-E heliks op die katalitiese aktiwiteit van die twee CYP11B1 isoforme is bepaal.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: • the comparison of the enzymatic activities of the two ovine cytochrome P450 17 - hydroxylase/17,20-lyase (CYP17) isoforms expressed in non-steroidogenic COS-1 cells. The Km and Vmax values for the metabolism of pregnenolone and progesterone were determined, while time-dependent metabolism of pregnenolone, 17-hydroxypregenolone, progesterone and 17-hydroxyprogesterone was also reported. The cloning and sequencing of ovine cytochrome b5 is reported and was co-expressed with CYP17. The results showed that the wild type 1 (WT1) isoform of ovine CYP17 produce more cortisol precursors than the wild type 2 (WT2) isoform; • the analysis of the frequency distribution of the CYP17 genotypes within a South African Merino population, which were divergently selected for (H-line) or against (L-line) the ability of a ewe to rear multiple offspring per birthing opportunity. It was observed that the CYP17 frequency distribution was the same within the H- and L-line, with 78.3 % heterozygous WT1/WT2 and 21.7 % homozygous WT1/WT1. No homozygous WT2/WT2 individuals were identified; • the development of a UPLC-MS/MS method for the separation and quantification of all thirteen adrenal steroids that are produced in the adrenal gland; • the relative contribution of the CYP17 genotypes in the total steroidogenic output in adult adrenocortical cells from the adrenal glands of H- and L-line sheep, with particular emphasis on cortisol production. The adrenocortical cells from the H-line sheep showed a marked higher cortisol production than the L-line, while adrenocortical cells from homozygous WT1/WT1 sheep also produced more cortisol than heterozygous WT1/WT2 sheep; • the blood cortisol responses upon the stimulation of the HPA axis by insulin induced hypoglycaemia of the H- and L-line sheep with known CYP17 genotypes. It was observed that the CYP17 genotype and selection line are important factors affecting the cortisol responses of sheep, where L-line heterozygous WT1/WT2 sheep showed the lowest cortisol response and glucose recovery; • the association of the CYP17 genotype with behavioural responses of H- and L-line sheep to flock isolation stress, as well as the association of the CYP17 genotype with ewe reproduction and lamb output. While reproduction seemed to be unaffected by the CYP17 genotype, the behavioural stress responses of sheep to flock isolation correlated with the CYP17 genotype, where the heterozygous WT1/WT2 genotype was associated with a wilder nature. / AFRIKAANSE OPSOMMING: Hierdie studie ondersoek: • die vergelyking van die ensiemaktiwiteite vir twee isoforme van skaap sitochroom P450 17 -hidroksilase/17,20-liase (CYP17), wat uitgedruk was in nie-steroïed genererende COS- 1 selle. Die Km and Vmax waardes was bepaal vir die metabolisme van pregnenoloon en progesteroon, terwyl die tyd-afhanklike metabolisme van pregnenoloon, 17- hidroksiepregnenoloon, progesteroon en 17-hidroksieprogesteroon ook gerapporteer word. Die klonering en volgorde bepaling van skaap sitochroom b5 was gedoen en gevolglik was sitochroom b5 saam met CYP17 uitgedruk in COS-1 selle. Die resultate het gewys dat wilde tipe 1 (WT1) meer voorlopers van kortisol produseer as wilde tipe 2 (WT2); • die frekwensie distrubusie van die CYP17 genotipes in ‘n Suid-Afrikaanse Merino populasie, waar skape in teenoorgestelde rigtings geselekteer was vir (H-lyn) of teen (L-lyn) die vermoë van ‘n ooi om geboorte te gee aan veelvoudige lammers per lamgeleentheid. Die frekwensie distrubusie van CYP17 was dieselfde in beide die H- en L-lyn, waar 78.3 % van die populasie heterosigoties WT1/WT2 en 21.7 % homosigoties WT1/WT1 was. Geen homosigote WT2/WT2 individue was geïdentifiseer nie; • die ontwikkeling van ‘n UPLC-MS/MS metode vir die skeiding en kwantifisering van al dertien steroïede wat natuurlik geproduseer word in die bynier van die skaap; • die relatiewe bydrae van die CYP17 isoforme tot die totale steroïedale uitsette vanuit die bynier kortex selle, vanaf die byniere van H- en L-lyn skape, waar klem geplaas word op die produksie van kortisol. Die bynierselle van die H-lyn skape het aansienlik meer kortisol produseer as die L-lyn, terwyl die bynierselle van die homosigotiese WT1/WT1 skape ook meer kortisol produseer het as heterosigotiese WT1/WT2 skape; • die bloed kortisol in reaksie tot die stimulering van die hipotalamus-hipofise-adrenale aksis, deur insulien geïnduseerde hipoglisemiese stress, in skape van die H- en L-lyne met bekende CYP17 genotipes. Dit was gevind dat die kortisol reaksie geaffekteer word deur beide die CYP17 genotipe en seleksie lyn, waar L-lyn heterosigotiese WT1/WT2 skape die minste kortisol geproduseer het en die stadigste herstel van glukose vlakke getoon het; • die assosiasie tussen die CYP17 genotipe en die gedrags reaksies op trop-isolasie, sowel as ooi-reproduksie en lamuitset, van die H- en L-lyn skape. Die reproduksie parameters was onafhanklik van die CYP17 genotipe, terwyl ‘n sterk assosiasie gevind was tussen die CYP17 genotipe en gedrags reaksies op trop-isolasie. Die heterosigotiese WT1/WT2 skape het ‘n wilder natuur getoon gedurende trop-isolasie in vergelyking met homosigotiese WT1/WT1 skape.

Saccharomyces cerevisiae tem sido empregada como modelo experimental em testes de mutagenicidade, devido à presença de um sistema citocromo P-450 capaz de metabolizar substâncias promutagênicas a sua forma ativa. O grande número de linhagens e as diferenças na capacidade de produção de citocromo P-450 dificultam a definição das condições ideais de cultivo para obtenção de células com alto conteúdo desta enzima, sendo que poucos são os trabalhos publicados a este respeito. O objetivo deste trabalho é avaliar as melhores condições de cultivo para produção de citocromo P-450 em S. cerevisiae. Para tanto, quatro diferentes linhagens foram cultivadas sob diferentes condições, a fim de avaliar a influência de fatores como fonte de carbono, tempo de incubação e adição de etanol ao meio de cultura. Foi verificado que células cultivadas na presença de fontes de carbono altamente fermentáveis e coletadas no final da fase de crescimento exponencial apresentavam o maior conteúdo da enzima. Observou-se também que o nível de citocromo P-450 variou entre as linhagens estudadas, bem como o tempo de incubação para atingir a concentração máxima, sendo que o conteúdo mais alto foi encontrado na linhagem ATCC 44953. A adição de 2 % de etanol (v/v) ao meio de cultura contendo 2 % de glicose (p/v) resultou em aumento do nível de citocromo P-450, indicando o efeito indutor do etanol. Os resultados deste trabalho mostram a importância de estabelecer condições de cultivo para obtenção de células com altas concentrações da enzima, uma vez que fatores como a composição do meio de cultura, o tempo de incubação e a linhagem empregada podem influenciar a produção de citocromo P-450 por S. cerevisiae. / Saccharomyces cerevisiae has been employed as experimental model in mutagenicity tests due to the presence of a cytochrome P-450 system capable of metabolizing promutagens to its active form. The large number of S.cerevisiae strains and their different capacity of producing cytochrome P-450 make the definition of the ideal cultivation conditions to obtain cells with high enzyme content difficult. Moreover, there are only a few reports related to this subject. The aim of this work is evaluate the ideal cultivation conditions to produce cytochrome P-450 by S. cerevisiae. For this purpose, four different S. cerevisiae strains were cultured under different cultivation conditions in order to evaluate the influence of factors such as carbon source, incubation time and addition of ethanol to the culture media. It was found that the cytochrome P-450 content was higher in cells growth on strongly fermentable carbon source and harvested at the end of the exponential growth phase. Our results also showed that the cytochrome P-450 concentration varied among the strains studied, as well as the incubation time to reach the maximum level. The highest content was found in the ATCC 44953 strain and the addition of 2 % ethanol (w/w) to the culture media containing 2 % glucose (w/v) increased the cytochrome P-450 amount, indicating the induction of cytochrome P-450 production by ethanol action. These results show the importance of establishment of the cultivation conditions to obtain cells with high cytochrome P-450 concentrations, considering that factors such as the culture media, incubation time and strain employed can influence the cytochrome P-450 production by S.cerevisiae.

Sun Huimin. / Thesis submitted in: December 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 77-89). / Abstracts in English and Chinese. / TITLE PAGE --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT IN CHINESE --- p.v / LIST OF PUBLICATIONS --- p.vii / ABBREVIATIONS --- p.viii / TABLE OF CONTENTS --- p.ix / Chapter CHAPTER 1. --- General Introduction --- p.1 / Chapter 1.1 --- Current Status of Herbal Product Use --- p.1 / Chapter 1.2 --- Herb-drug interactions --- p.2 / Chapter 1.2.1. --- Mechanisms of herb-drug interaction --- p.3 / Chapter 1.2.2. --- Pharmacodynamic interaction --- p.3 / Chapter 1.2.3. --- Pharmacokinetic interaction --- p.4 / Chapter 1.2.4. --- Herb-drug interaction involving drug metabolizing enzymes --- p.5 / Chapter 1.3 --- Methodologies for studying herb-drug interactions involving CYP enzymes --- p.7 / Chapter 1.3.1. --- Animal studies (Ex vivo approach) --- p.7 / Chapter 1.3.2. --- In vitro inhibition/induction studies --- p.8 / Chapter 1.3.3. --- Clinical studies --- p.9 / Chapter CHAPTER 2. --- Effect of flavonoid-containing herbs on CYP 450 enzyme activities: a screening study in rat --- p.11 / Chapter 2.1 --- Introduction --- p.11 / Chapter 2.2 --- Materials and Methods --- p.12 / Chapter 2.2.1. --- Chemicals --- p.12 / Chapter 2.2.2. --- Herbs --- p.12 / Chapter 2.2.3. --- Preparation of herbal extracts --- p.13 / Chapter 2.2.3.1. --- Preparation of Green Tea extract --- p.13 / Chapter 2.2.3.2. --- Preparation of Decaffeinated Green Tea (DGT) and its extracts --- p.13 / Chapter 2.2.3.3. --- "Preparation of extracts of Huang Qin, Ge Gen and Huai Mi" --- p.14 / Chapter 2.2.3.4. --- Preparation of Ginkgo biloba extract suspension --- p.14 / Chapter 2.2.4. --- Animal treatment --- p.14 / Chapter 2.2.5. --- Preparation of rat liver microsomes --- p.15 / Chapter 2.2.6. --- Determination of protein content of liver microsomes --- p.16 / Chapter 2.2.7. --- Determination of microsomal CYP content --- p.17 / Chapter 2.2.8. --- Statistical analysis --- p.19 / Chapter 2.3 --- Results --- p.19 / Chapter 2.4 --- Discussion --- p.24 / Chapter 2.5 --- Conclusion --- p.25 / Chapter CHAPTER 3 --- Rationale of the clinical study --- p.26 / Chapter CHAPTER 4 --- Development of HPLC methods for simultaneous determination of multiple probe drugs and their metabolites in human plasma or urine --- p.30 / Chapter 4.1 --- Introduction --- p.30 / Chapter 4.2 --- Materials and Methods --- p.33 / Chapter 4.2.1. --- Chemicals and reagents --- p.33 / Chapter 4.2.2. --- Preparation of stock and working solutions --- p.33 / Chapter 4.2.3. --- Equipment and chromatographic conditions --- p.34 / Chapter 4.2.4. --- Treatment of plasma and urine samples with β-glucuronidase --- p.35 / Chapter 4.2.5. --- Extraction procedures --- p.36 / Chapter 4.2.6. --- Preparation of working solutions for calibration curve --- p.37 / Chapter 4.3 --- Results --- p.39 / Chapter 4.3.1. --- Separation of the analytes --- p.39 / Chapter 4.3.2. --- Calibration and linearity --- p.39 / Chapter 4.3.3. --- Sensitivity --- p.39 / Chapter 4.3.4 --- Accuracy and precision --- p.50 / Chapter 4.4 --- Discussion --- p.52 / Chapter 4.5 --- Conclusions --- p.53 / Chapter CHAPTER 5 --- Stability study of probe drugs --- p.54 / Chapter 5.1 --- Introduction --- p.54 / Chapter 5.2 --- Materials and Methods --- p.54 / Chapter 5.2.1. --- Preparation of standard solutions of probe drugs --- p.54 / Chapter 5.2.2. --- Preparation of stability study mediums --- p.54 / Chapter 5.2.2.1. --- Gastric juice (pH=1.2) --- p.54 / Chapter 5.2.2.2. --- Intestine fluid (pH=6.8) --- p.54 / Chapter 5.2.2.3. --- Human plasma (pH=7.4) --- p.55 / Chapter 5.2.2.4. --- Phosphate buffer (pH=7.4) --- p.55 / Chapter 5.2.3. --- Incubation --- p.55 / Chapter 5.2.4. --- Determination of probe drug concentrations in incubation samples --- p.56 / Chapter 5.3 --- Results --- p.57 / Chapter 5.4 --- Discussion --- p.59 / Chapter 5.5 --- Conclusion --- p.59 / Chapter CHAPTER 6 --- Effect of the extract of Ginkgo biloba leaf (761) on CYP isozymes in human subjects --- p.60 / Chapter 6.1 --- Introduction --- p.60 / Chapter 6.2 --- Materials and Methods --- p.60 / Chapter 6.2.1. --- Drugs --- p.60 / Chapter 6.2.2. --- Subjects --- p.61 / Chapter 6.2.3. --- Study design --- p.62 / Chapter 6.2.4. --- Determination of probe drugs/metabolites in the plasma and urine --- p.63 / Chapter 6.2.5. --- Data analysis --- p.65 / Chapter 6.2.6. --- Statistical analysis --- p.66 / Chapter 6.3 --- Results --- p.66 / Chapter 6.3.1. --- Effect of EGb761on CYP1A2 activity --- p.66 / Chapter 6.3.2. --- Effect of EGb761on CYP2E1 activity --- p.67 / Chapter 6.3.3. --- Effect of EGb761 on CYP450 3A activity --- p.68 / Chapter 6.3.4. --- Effect of EGb761 on NAT2 activity --- p.69 / Chapter 6.3.5. --- Effect of EGb761 on CYP2D6 activity --- p.70 / Chapter 6.3.6. --- Effects of EGb761 on CYP2C19 activity --- p.71 / Chapter 6.4 --- Discussion --- p.72 / Chapter 6.5 --- Conclusion --- p.76 / References --- p.77 / Appendix --- p.90