Luffa operculata é uma planta medicinal popularmente usada para tratamento de rinites e rinossinusites. A infusão de seu fruto seco é usada no nariz, liberando secreção mucosa profusa, mas pode causar irritação, epistaxe ou anosmia. Avaliamos os efeitos de diferentes concentrações da infusão do fruto seco da Luffa operculata na velocidade de transporte mucociliar (VTM), freqüência de batimento ciliar (FBC), diferença de potencial transepitelial (DPT) e morfologia do epitélio (microscopia de luz e eletrônica de transmissão), no modelo do palato isolado de rã. Os resultados apontam para dano epitelial dose-dependente no epitélio mucociliar, sugerindo que ela seja potencialmente nociva à mucosa nasal humana / Luffa operculata is a medicinal plant popularly used for treatment of rhinitis and rhinosinusitis. Its dry fruit infusion is used into the nose, delivering profuse mucous secretion, but may cause nasal mucosa irritation, epistaxis or anosmia. We evaluated the effects of different concentrations of Luffa operculata dry fruit infusion on mucociliary transport velocity (MTV), ciliary beat frequency (CBF), transepithelial potential difference (TPD) and epithelial morphology (light and electron transmission microscopy) of the isolated frog palate preparation. Results pointed to dose-dependent epithelial damage on mucociliary epithelium, suggesting that it is potentially noxious to the human nasal mucosa

by Wai Ching Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 154-159). / Abstracts in English and Chinese. / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / List of Abbreviations --- p.viii / Table of contents --- p.x / List of Tables --- p.xiv / List of Figures --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.4 / Chapter 2.1 --- Plant lectins --- p.4 / Chapter 2.1.1 --- Introduction --- p.4 / Chapter 2.1.2 --- Definition and subdivision of plant lectins --- p.4 / Chapter 2.2 --- Monocot mannose-binding lectins --- p.6 / Chapter 2.2.1 --- Occurrence and carbohydrate binding specificity --- p.6 / Chapter 2.2.2 --- Molecular structure and amino acid sequence --- p.7 / Chapter 2.2.3 --- "Molecular cloning, biosynthesis and post-translational modification" --- p.10 / Chapter 2.2.4 --- Mannose-binding lectins of Family Liliaceae --- p.11 / Chapter 2.2.4.1 --- Tulipa gesneriana lectins (TGL) --- p.12 / Chapter 2.2.4.2 --- Aloe arborescens lectins (AAL) --- p.13 / Chapter 2.2.4.3 --- Polygonatum multiflorum agglutinin (PMA) and lectin-related protein --- p.14 / Chapter 2.3 --- Polygonatum odoratum lectins (POL) --- p.15 / Chapter 2.3.1 --- Isolation and purification of POL from Yu Chu --- p.15 / Chapter 2.3.2 --- Agglutinating activity and anti-viral activities of POL --- p.17 / Chapter 2.3.3 --- Bacterial expression of POL in Escherichia coli --- p.18 / Chapter 2.4 --- Plant-based production of recombinant proteins --- p.20 / Chapter 2.4.1 --- Advantages of using plants as expression system --- p.20 / Chapter 2.4.2 --- Plant-derived recombinant proteins --- p.22 / Chapter 2.5 --- Expression of heterologous proteins in rice --- p.24 / Chapter 2.5.1 --- The facts of rice --- p.24 / Chapter 2.5.2 --- Rice storage proteins --- p.25 / Chapter 2.5.2 --- Expression of lysine-rich protein (LRP)/glutelin fusion proteinin rice seeds --- p.28 / Chapter 2.5.3 --- Expression of Galanthus nivalis agglutinin in rice --- p.29 / Chapter 2.6 --- Protein trafficking in plants --- p.30 / Chapter 2.6.1 --- Golgi-dependent pathways --- p.30 / Chapter 2.6.2 --- Golgi-independent pathway --- p.32 / Chapter 2.6.3 --- Expression of protein targeting determinants in tobacco plants and suspension cells --- p.33 / Chapter Chapter 3 --- Materials and Methods --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Chemcials --- p.35 / Chapter 3.3 --- Bacterial strains --- p.35 / Chapter 3.4 --- Cloning of POL cDNA --- p.36 / Chapter 3.4.1 --- Plant materials --- p.36 / Chapter 3.4.2 --- RNA extraction --- p.36 / Chapter 3.4.3 --- RT-PCR amplification of POL cDNA --- p.36 / Chapter 3.4.4 --- 5'RACE and 3'RACE --- p.38 / Chapter 3.4.5 --- Sequencing of POL cDNA --- p.39 / Chapter 3.5 --- Analysis of POL protein --- p.40 / Chapter 3.5.1 --- Protein extraction and Tricine-SDS PAGE --- p.40 / Chapter 3.5.2 --- Western blot analysis --- p.41 / Chapter 3.6 --- Chimeric gene construction --- p.42 / Chapter 3.6.1 --- Construction of the Cauliflower mosaic virus (CaMV)35S promoter/POL constructs --- p.44 / Chapter 3.6.2 --- Construction of the glutelin-1 promoter/POL constructs --- p.48 / Chapter 3.6.3 --- Sequence fidelity of chimeric genes --- p.55 / Chapter 3.7 --- Expression of transgenes in rice --- p.55 / Chapter 3.7.1 --- Plant materials --- p.55 / Chapter 3.7.2 --- Agrobacterium transformation --- p.55 / Chapter 3.7.3 --- Callus induction --- p.56 / Chapter 3.7.4 --- Agrobacterium culture and rice transformation --- p.56 / Chapter 3.7.5 --- Selection and regeneration of rice callus --- p.56 / Chapter 3.7.6 --- Isolation of genomic DNA --- p.58 / Chapter 3.7.7 --- Southern blot analysis --- p.58 / Chapter 3.7.8 --- Extraction of leaf total RNA --- p.59 / Chapter 3.7.9 --- Extraction of seed total RNA --- p.59 / Chapter 3.7.10 --- Northern blot analysis --- p.60 / Chapter 3.7.11 --- Protein extraction and Tricine SDS-PAGE --- p.60 / Chapter 3.7.12 --- Western blot analysis --- p.61 / Chapter 3.8 --- Cytopathic effect (CPE) reduction assay --- p.61 / Chapter 3.8.1 --- Protein extraction --- p.61 / Chapter 3.8.2 --- CPE reduction assay --- p.62 / Chapter 3.9 --- Confocal immunofluorescence --- p.63 / Chapter 3.9.1 --- Preparation of sections --- p.63 / Chapter 3.9.2 --- Labelling of fluorescence probes --- p.63 / Chapter 3.9.3 --- Image collection --- p.64 / Chapter Chapter 4 --- Results --- p.65 / Chapter 4.1 --- Cloning of POL cDNA from Yu Chu --- p.65 / Chapter 4.1.1 --- RNA extraction and partial POL cDNA amplification --- p.65 / Chapter 4.1.2 --- 5'RACE and 3'RACE --- p.67 / Chapter 4.1.3 --- Sequencing of POL cDNA --- p.68 / Chapter 4.1.4 --- Sequences comparison of POL and Liliaceae lectins --- p.75 / Chapter 4.2 --- Occurence of POL protein in Yu Chu plant --- p.77 / Chapter 4.3 --- Constitutional expression of POL in rice --- p.79 / Chapter 4.3.1 --- Construction of Cauliflower mosaic virus 35S promoter constructs --- p.80 / Chapter 4.3.2 --- Southern blot analysis --- p.82 / Chapter 4.3.3 --- Northern blot analysis --- p.84 / Chapter 4.3.4 --- Western blot analysis --- p.85 / Chapter 4.3.5 --- Western blot analysis of 35S/POL T1 plant --- p.87 / Chapter 4.4 --- Seed-specific expression of POL in rice --- p.88 / Chapter 4.4.1 --- Construction of the glutelin-1 promoter constructs --- p.89 / Chapter 4.4.2 --- Southern blot analysis --- p.92 / Chapter 4.4.3 --- Northern blot analysis --- p.96 / Chapter 4.4.4 --- Western blot analysis --- p.101 / Chapter 4.4.5 --- Western blot analysis of POL-BP-8O and POL-α-TIP T1 transgenic plants --- p.117 / Chapter 4.5 --- Cytopathic effect (CPE) reduction assay --- p.122 / Chapter 4.6 --- Confocal immunofluorescence studies --- p.125 / Chapter Chapter 5 --- Discussion --- p.134 / Chapter 5.1 --- Cloning of POL cDNA --- p.134 / Chapter 5.2 --- Analysis of constitutional expression of POL in rice --- p.136 / Chapter 5.3 --- Analysis of seed-specific expression of POL in rice --- p.138 / Chapter 5.4 --- Localization of POL in POL-BP-8O and POL-α-TIP transgenic rice seeds --- p.146 / Chapter 5.5 --- Cytopathic effect (CPE) reduction assay --- p.148 / Chapter 5.6 --- Future prospects --- p.151 / Chapter Chapter 6 --- Conclusion --- p.153

Luffa operculata é uma planta medicinal popularmente usada para tratamento de rinites e rinossinusites. A infusão de seu fruto seco é usada no nariz, liberando secreção mucosa profusa, mas pode causar irritação, epistaxe ou anosmia. Avaliamos os efeitos de diferentes concentrações da infusão do fruto seco da Luffa operculata na velocidade de transporte mucociliar (VTM), freqüência de batimento ciliar (FBC), diferença de potencial transepitelial (DPT) e morfologia do epitélio (microscopia de luz e eletrônica de transmissão), no modelo do palato isolado de rã. Os resultados apontam para dano epitelial dose-dependente no epitélio mucociliar, sugerindo que ela seja potencialmente nociva à mucosa nasal humana / Luffa operculata is a medicinal plant popularly used for treatment of rhinitis and rhinosinusitis. Its dry fruit infusion is used into the nose, delivering profuse mucous secretion, but may cause nasal mucosa irritation, epistaxis or anosmia. We evaluated the effects of different concentrations of Luffa operculata dry fruit infusion on mucociliary transport velocity (MTV), ciliary beat frequency (CBF), transepithelial potential difference (TPD) and epithelial morphology (light and electron transmission microscopy) of the isolated frog palate preparation. Results pointed to dose-dependent epithelial damage on mucociliary epithelium, suggesting that it is potentially noxious to the human nasal mucosa

<p>Ruzmarin Rosmarinus officinalis L. (Lamiaceae) je biljka koja se u tradicionalnoj medicini na na&scaron;em području koristi za postizanje analgetičkog, holeretičkog i hepatoprotektivnog delovanja. Prema Evropskoj agenciji za lekove (2010 godine), indikacije za sistemsku primenu etarskog ulja ruzmarina su lečenje dispepsije i spazama gastrointestinalnog trakta, a za spolja&scaron;nju primenu se preporučuje u lečenju umereno jakih bolova u zglobovima i mi&scaron;ićima i u lečenju poremećaja periferne cirkulacije. Imajući u vidu da komponente etarskog ulja ruzmarina ispoljavaju i druga, potencijalno korisna farmakolo&scaron;ka svojstva, postoji potreba da se ova delovanja detaljnije ispitaju. Ciljevi ispitivanja su bili da se utvrdi: 1) analgetički efekat etarskog ulja ruzmarina i njegov uticaj na farmakodinamske osobine paracetamola, kodeina, diazepama i pentobarbitala kao i na farmakokinetske osobine paracetamola; 2) antioksidativni i hepatoprotektivni efekat u uslovima hemijski izazvanog oksidativnog stresa. Metodom gasne hromatografije (GC/MS i GC/FID) utvrđen je kvantitativni sastav etarskog ulja. Najzastupljenije komponente ulja koje je kori&scaron;ćeno u na&scaron;em ispitivanju su oksidovani monoterpeni 1,8-cineol (43.77%) i kamfor (12.53%) i monoterpenski ugljovodonik &alpha;-pinen (11.51%). Suspenzija etarskog ulja ruzmarina primenjivana je mi&scaron;evima u dozama 10 i 20 mg/kg tm tokom sedam dana i jednokratno u farmakodinamskim testovima: test vrele ploče, test &bdquo;uvijanja&ldquo; (posle intraperitonealne primene sirćetne kiseline), test za procenu motorne koordinacije životinja na rotirajućem &scaron;tapu i test merenja vremena trajanja spavanja. Za ispitivanje uticaja etarskog ulja ruzmarina na farmakokinetske osobine paracetamola i za biohemijska i toksikolo&scaron;ka ispitivanja, kori&scaron;ćeni su pacovi koji su tokom sedam dana tretirani suspenzijom etarskog ulja ruzmarina u dozi 5 i 10 mg/kg tm, a sedmog dana su primili paracetamol i.v. ili p.o.. Za praćenje farmakokinetskih parametara kori&scaron;ćeni su uzorci krvi dobijeni iz repne vene pacova u kojima su HPLC metodom merene koncentracije paracetamola, na osnovu kojih su potom određeni farmakokinetski parametri ovog leka. Antioksidativna aktivnost etarskog ulja ruzmarina je određivana in vitro (DPPH i Folin-Ciocaulteu testovima) i in vivo. Nakon žrtvovanja životinja iz prikupljenih uzoraka krvi određivani su iz seruma biohemijski parametri, pokazatelji bubrežne i jetrene funkcije, a u homogenatu tkiva jetre određivani su parametri oksidativnog stresa. Samo etarsko ulje ruzmarina ispoljava analgetičko delovanje i smanjuje visceralnu bol izazvanu sirćetnom kiselinom. Pored toga, potencira analgetički efekat kodeina i paracetamola. Etarsko ulje ruzmarina značajno smanjuje hipnotičko delovanje pentobarbitala i sprečava poremećaj motorne koordinacije nakon primene diazepama. Etarsko ulje ruzmarina ne utiče značajnije na oralnu biolo&scaron;ku raspoloživost paracetamola. Vi&scaron;ekratna primena različitih doza etarskog ulja ruzmarina ne izaziva toksične promene u krvi i jetri ispitivanih životinja. Primena etarskog ulja ruzmarina &scaron;titi životinje od reaktivnih kiseoničnih vrsta, umanjuje posledice izloženosti oksidativnom stresu i ispoljava značajno hepatoprotektivno delovanje.</p> / <p>Rosemary Rosmarinus officinalis, L.(Lamiaceae) is traditionally used in folk medicine for its analgetic, choleretic and hepatoprotective properties. According to the recommendation of European Medicines Agency from 2010, rosemary essential oil can be used for treating dyspepsia and mild spasmodic disorders of the gastrointestinal tract, and also externally as an adjuvant in the relief of minor muscular and articular pain and minor peripheral circulatory disorders. Different studies conducted with rosemary essential oil show other pharmacological effects of main components of the oil. The aim of this study was to examine: 1) analgetic effects of rosemary essential oil and its influence on the pharmacodynamic properties of paracetamol, codeine, diazepam and pentobarbital, and also its influence on the pharmacokinetic properties of paracetamol; 2) antioxidant and hepatoprotective effects on the parameters of chemicaly induced oxidative stress. The quantification of chemical constituents of the essential oil was carried out by gas chromatography (GC/FID and GC/MS). The major compounds that were identified and quantitated by GC-FID and GC-MS were oxygenated monoterpens 1,8-cineole (43.77%), camphor (12.53%) and monoterpene hydrocarbon &alpha;-pinene (11.51%). The suspension of rosemary essential oil was applied to mice orally (doses: 10 and 20 mg/kg b.w.) for seven days and in single dose for the pharmacodynamic tests: hot plate, writhing, rotharod and sleeping time. Rats treated with suspension of rosemary essential oil for seven days orally (doses: 5 and 10 mg/kg b.w.) were used for the examination of influence of essential oil on the pharmacokinetic properties of paracetamol. Then on the 7th day the paracetamol was applied to them p.o. or i.v.. The parameters of pharmacokinetic were analyzed in blood samples obtained from rats tail veins. The HPLC method was used for measurement of concentration of paracetamol in blood samples. Those concentrations were used for calculation of the pharmacokinetic parameters. The antioxidant activity of the rosemary essential oil was evaluated in vitro (with DPPH and Folin-Ciocaulteu tests) and in vivo. The animals were sacrificed and the samples of blood and liver were taken. The obtained serum was used for determination of standard biochemical parameters and the parameters of oxidative stress were analyzed in obtained liver homogenates. The essential oil of rosemary shows analgetic properties and it decreases visceral pain induced with intraperitoneally injected acetic acid. The rosemary essential oil increases pharmacological effects of codeine and paracetamol. Also, this oil reduces pentobarbital-induced sleeping time and diminishes diazepam-induced disorder of psychomotor coordination. The essential oil of rosemary does not change paracetamol bioavailability. The rosemary essential oil applied in multiple doses does not induce toxic changes in blood and liver samples obtained from animals. The use of rosemary essential oil protects animals from reactive oxygen species, decreases the effects caused by oxidative stress and shows significant hepatoprotective effect.</p>

by Lam Tin Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 131-151). / Abstracts in English and Chinese. / Acknowledgment --- p.I / Table of content --- p.II / List of Tables --- p.IX / List of Figures --- p.XI / Abbreviation --- p.XIII / Abstract --- p.XIV / 論文摘要 --- p.XVI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Acquired immunodeficiency syndrome (AIDS) --- p.1 / Chapter 1.1.1 --- History of AIDS --- p.1 / Chapter 1.1.2 --- Definition of AIDS --- p.2 / Chapter 1.1.3 --- HIV/AIDS Around the World --- p.4 / Chapter 1.1.4 --- HIV/AIDS in Hong Kong --- p.4 / Chapter 1.1.4.1 --- Hong Kong AIDS Update --- p.4 / Chapter 1.1.4.2 --- AIDS Transmission --- p.6 / Chapter 1.1.4.3 --- Main AIDS Complications Occur in Hong Kong --- p.6 / Chapter 1.2 --- Human Immunodeficiency Virus (HIV) --- p.7 / Chapter 1.2.1 --- Classification of HIV --- p.7 / Chapter 1.2.2 --- The Structure of HIV Virion --- p.9 / Chapter 1.2.3 --- The HIV Genome --- p.11 / Chapter 1.2.4 --- The Life Cycle of HIV --- p.12 / Chapter 1.2.4.1 --- Invasion of the Cells --- p.12 / Chapter 1.2.4.2 --- Integration into cell genome --- p.13 / Chapter 1.2.4.3 --- Protease and assembly to the virus --- p.13 / Chapter 1.2.5 --- Three Essential Enzymes for HTV-1 Replication --- p.16 / Chapter 1.2.5.1 --- HIV-1 Reverse Transcriptase (HIV-1 RT) --- p.16 / Chapter 1.2.5.2 --- HIV-1 Integrase (HIV-1 IN) --- p.17 / Chapter 1.2.5.3 --- HIV-1 Protease (HIV-1 PR) --- p.18 / Chapter 1.2.6 --- The Different Stages of HIV Infection --- p.19 / Chapter 1.3 --- AIDS therapy --- p.23 / Chapter 1.3.1 --- Drugs Approved by US Food and Drug Administration (FDA) --- p.23 / Chapter 1.3.2 --- Vaccine --- p.26 / Chapter 1.3.3 --- Chemokine Receptor Inhibitor --- p.27 / Chapter 1.3.4 --- Antisense Oligonucleotides Therpay --- p.28 / Chapter 1.3.5 --- Traditional Chinese Medicine (TCM) --- p.29 / Chapter 1.4 --- Objective of My Project --- p.32 / Chapter CHAPTER 2 --- SCREENING OF TRADITIONAL CHINESE MEDICINAL PLANTS FOR HIV-1 PROTEASE INHIBITION --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and Methods --- p.35 / Chapter 2.2.1 --- Materials --- p.35 / Chapter 2.2.2 --- Extraction Methods --- p.36 / Chapter 2.2.2.1 --- Aqueous Extraction --- p.36 / Chapter 2.2.2.2 --- Methanol Extraction --- p.37 / Chapter 2.2.3 --- Preparation of Recombinant HIV-1 Protease --- p.37 / Chapter 2.2.3.1 --- Selection of Appropriate Clone --- p.37 / Chapter 2.2.3.2 --- Large-scale Expression of Recombinant HIV-1 Protease --- p.38 / Chapter 2.2.2.3 --- Purification of Recombinant HIV-1 Protease by DEAE Sepharose CL-6B Chromatography --- p.38 / Chapter 2.2.3.4 --- Purification of Recombinant HIV-1 Protease by Mono-S Cation Chromatography --- p.39 / Chapter 2.2.3.5 --- Refolding of Purified Recombinant HIV-1 Protease --- p.40 / Chapter 2.2.3.6 --- Protein Concentration Determination --- p.41 / Chapter 2.2.3.7 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.41 / Chapter 2.2.4 --- Characterization of HTV-1 Protease --- p.42 / Chapter 2.2.4.1 --- HIV-1 PR Fluorogenic Assays --- p.42 / Chapter 2.2.4.2 --- HIV-1 PR Assay by Reverse Phase HPLC Separation of Cleavage Products of the Synthetic Peptide Substrate --- p.43 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- Functional Analysis of Recombinant HIV-1 PR Activity --- p.44 / Chapter 2.3.2 --- Screening of Crude Extracts for Inhibition of HIV-1 PR Activity --- p.48 / Chapter 2.4 --- Discussion --- p.53 / Chapter CHAPTER 3 --- ISOLATION AND CHARACTERIZATION OF ACTIVE CONSTITUENTS FROM METHANOL EXTRACTS OF WOODWARDIA UNIGEMMATA AGAINST HIV-1 PROTEASE --- p.56 / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.57 / Chapter 3.2.1 --- Materials --- p.57 / Chapter 3.2.2 --- Methods --- p.58 / Chapter 3.2.2.1 --- Methanol Extraction --- p.58 / Chapter 3.2.2.2 --- Removal of Tannins --- p.60 / Chapter 3.2.2.3 --- Glucosidase Digestion --- p.60 / Chapter 3.2.2.4 --- Analytical Thin Layer Chromatographic (TLC) --- p.61 / Chapter 3.2.2.5 --- A cid Hydrolysis --- p.62 / Chapter 3.2.2.6 --- Electrospray Mass Spectrometry --- p.62 / Chapter 3.2.2.7 --- Dose-response Curve --- p.63 / Chapter 3.2.2.8 --- Kinetic Studies --- p.63 / Chapter 3.2.2.9 --- Activity of the HPLC-purified principle (s) on Other Aspartyl Proteases --- p.63 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Purification of Methanol Extracts of Woocdwardia unigemmata --- p.66 / Chapter 3.2.2 --- Removal of Tannins --- p.70 / Chapter 3.2.3 --- Glucosidase Digestion --- p.73 / Chapter 3.2.4 --- Acid Hydrolysis --- p.73 / Chapter 3.2.5 --- Analytical Thin Layer Chromatography --- p.74 / Chapter 3.2.6 --- Electrospray Mass Spectrometry --- p.80 / Chapter 3.2.7 --- Dose-response Inhibition of HIV-1 Protease --- p.80 / Chapter 3.2.8 --- Kinetic Studies --- p.85 / Chapter 3.2.9 --- Effects of HPLC-purified Active Principle on Other Aspartyl Proteases --- p.87 / Chapter 3.3 --- Discussion --- p.89 / Chapter CHATPER 4 --- IDENTIFICATION OF SELECTIVE RNA APTAMERS AGAINST HIV-1 PROTEASE BY SYSTEMATIC EVOLUTION OF LIGANDS BY EXPONENTIAL ENRICHMENT (SELEX) --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Materials and Methods --- p.101 / Chapter 4.2.1 --- Materials --- p.101 / Chapter 4.2.2 --- Methods --- p.102 / Chapter 4.2.2.1 --- PCR Amplification for the Generation of a Double-Stranded DNA Library --- p.103 / Chapter 4.2.2.2 --- Preparation of RNA Pools --- p.104 / Chapter 4.2.2.3 --- In vitro Selection of RNA Ligands --- p.104 / Chapter 4.2.2.4 --- Reverse Transcription Reaction of Selected RNA --- p.108 / Chapter 4.2.2.5 --- Cloning of the Amplified cDNA pools --- p.108 / Chapter 4.2.2.6 --- Subcloning of the digested DNA product into pBluescript® IIKS (-) --- p.108 / Chapter 4.2.2.8 --- RNA Labeling with Digoxigenin (DIG) --- p.109 / Chapter 4.2.2.9 --- Binding Affinity of RNA Ligands for HIV-1 PR --- p.109 / Chapter 4.2.2.10 --- Competition Binding Reactions --- p.111 / Chapter 4.2.2.11 --- HIV-1 PR Inhibitory Activities of the Selected RNA Ligands --- p.112 / Chapter 4.3 --- Results --- p.113 / Chapter 4.3.1 --- In Vitro Selection of RNA Ligands --- p.113 / Chapter 4.3.2 --- Sequences of RNA Ligands --- p.114 / Chapter 4.3.3 --- Binding Affinity of RNA Ligands --- p.114 / Chapter 4.3.4 --- Inhibitory Activity of RNA Ligands --- p.119 / Chapter 4.4 --- Discussion --- p.122 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.128 / REFERENCES --- p.132

by Lam Mei Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 127-137). / Abstract also in Chinese. / Acknowledgment --- p.I / Table of content --- p.II / List of figures --- p.VII / List of tables --- p.IX / Abbreviation --- p.X / Abstract --- p.XII / 論文摘要 --- p.XIII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Acquired immunodeficiency syndrome --- p.1 / Chapter 1.1.1 --- Discovery of AIDS --- p.1 / Chapter 1.1.2 --- Definition and symptoms of AIDS --- p.1 / Chapter 1.1.3 --- AIDS transmission --- p.2 / Chapter 1.1.4 --- AIDS epidemic --- p.3 / Chapter 1.2 --- Human immunodeficiency virus --- p.3 / Chapter 1.2.1 --- Discovery of HIV --- p.3 / Chapter 1.2.2 --- The structure of HIV --- p.4 / Chapter 1.2.3 --- Genomic structure of HIV --- p.5 / Chapter 1.2.4 --- Life cycle of HIV --- p.5 / Chapter 1.2.5 --- How HIV is involved in different stages of AIDS --- p.7 / Chapter 1.3 --- Therapeutic targets for treatment of AIDS --- p.8 / Chapter 1.3.1 --- HIV reverse transcriptase (HIV RT) --- p.8 / Chapter 1.3.2 --- HIV integrase (HIV IN) --- p.11 / Chapter 1.3.3 --- HIV protease (HIV PR) --- p.12 / Chapter 1.3.4 --- Chemokine receptors --- p.14 / Chapter 1.3.5 --- Vaccine development --- p.16 / Chapter 1.4 --- AIDS therapy --- p.17 / Chapter 1.4.1 --- Current status of AIDS therapy --- p.17 / Chapter 1.4.1.1 --- Drugs approved by US Food & Drug Administration (FDA) --- p.17 / Chapter 1.4.1.2 --- Combination therapy --- p.19 / Chapter 1.4.1.3 --- Vaccine development --- p.19 / Chapter 1.4.2 --- Alternative treatment --- p.20 / Chapter 1.5 --- Objective of my project --- p.21 / Chapter Chapter 2 --- Screening of traditional Chinese medicinal (TCM) plants for HIV reverse transcriptase inhibition --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.1.1 --- HIV RT structure and function --- p.22 / Chapter 2.1.2 --- Natural product against HIV RT --- p.25 / Chapter 2.1.3 --- Inhibitory activities from plant extracts --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Extraction methods --- p.30 / Chapter 2.2.2.1 --- Methanol extraction --- p.30 / Chapter 2.2.2.2 --- Hot water extraction --- p.30 / Chapter 2.2.2.3 --- Preparation of Prunella vulgaris extract --- p.30 / Chapter 2.2.3 --- Reverse transcriptase assay --- p.31 / Chapter 2.2.4 --- Characterization of active component in extract of Prunella vulgaris --- p.32 / Chapter 2.2.4.1 --- Protease digestion --- p.32 / Chapter 2.2.4.2 --- Glucosidase digestion --- p.32 / Chapter 2.2.4.3 --- Ethanol precipitation --- p.33 / Chapter 2.2.4.4 --- Sodium periodiate oxidization --- p.33 / Chapter 2.2.4.5 --- Polyvinylpyrrolidone (PVP) Precipitation --- p.34 / Chapter 2.2.4.6 --- Polyamide resin binding --- p.34 / Chapter 2.2.5 --- Purification of Prunella vulgaris extract --- p.34 / Chapter 2.2.5.1 --- Polyamide resin column chromatography --- p.34 / Chapter 2.2.5.2 --- Sephadex LH-20 chromatography --- p.35 / Chapter 2.2.5.3 --- Reverse phase HPLC chromatography --- p.36 / Chapter 2.2.6 --- Characterization of purified Prunella vulgaris extract --- p.37 / Chapter 2.2.6.1 --- Paper chromatography --- p.37 / Chapter 2.2.6.2 --- Acid hydrolysis of extract --- p.37 / Chapter 2.2.6.3 --- Thin layer chromatography --- p.38 / Chapter 2.2.6.4 --- Other assays --- p.39 / Chapter 2.2.7 --- Calculation --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Screening of Herbs --- p.41 / Chapter 2.3.1.1 --- Screening of methanol extracts --- p.41 / Chapter 2.3.1.2 --- Screening of hot water extracts --- p.41 / Chapter 2.3.2 --- Characterization of active components in Prunella vulgaris crude extracts --- p.44 / Chapter 2.3.2.1 --- Protease digestion --- p.44 / Chapter 2.3.2.2 --- Glucosidase digestion --- p.44 / Chapter 2.3.2.3 --- Ethanol precipitation --- p.44 / Chapter 2.3.2.4 --- Sodium periodate oxidation --- p.48 / Chapter 2.3.2.5 --- Effect of naturally occurring chemicals on inhibition of HIV RT --- p.48 / Chapter 2.3.2.6 --- Effect of removal of polyphenolic components of aqueous extract on inhibition of HTV RT --- p.51 / Chapter 2.3.3 --- Further purification of active components in aqueous extract of Prunella vulgaris --- p.53 / Chapter 2.3.3.1 --- Absorption chromatography by polyamide resin --- p.53 / Chapter 2.3.3.2 --- The Sephadex LH-20 chromatography --- p.53 / Chapter 2.3.3.3 --- Reverse phase high performance liquid chromatography --- p.56 / Chapter 2.3.3.4 --- Recovery of extract --- p.59 / Chapter 2.3.3.5 --- Inhibition from extract of various steps of purification --- p.59 / Chapter 2.3.4 --- Characterization of purified aqueous extract of Prunella vulgaris --- p.62 / Chapter 2.3.4.1 --- Paper chromatography --- p.62 / Chapter 2.3.4.2 --- Dose response curve --- p.62 / Chapter 2.3.4.3 --- Acid hydrolysis of purified extract --- p.68 / Chapter 2.3.4.4 --- Identification of monosaccharide in purified extract by Thin layer chromatography (TLC) --- p.71 / Chapter 2.3.5 --- Specificity of the purified extract on polymerase inhibition --- p.75 / Chapter 2.3.5.1 --- Inhibition of purified Prunella vulgaris extract on Taq polymerase --- p.75 / Chapter 2.3.5.2 --- Inhibition of purified Prunella vulgaris extract on Superscript II --- p.75 / Chapter 2.4 --- Discussion --- p.79 / Chapter Chapter 3 --- Screening of inhibitory activities from traditional Chinese medicinal (TCM) plants extracts to HIV protease --- p.86 / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- HIV Protease structure and function --- p.86 / Chapter 3.1.2 --- Natural products against HIV Protease --- p.87 / Chapter 3.1.3 --- Plant extracts against HIV Protease --- p.89 / Chapter 3.2 --- Materials and Methods --- p.91 / Chapter 3.2.1 --- Materials --- p.91 / Chapter 3.2.2 --- Expression of HIV protease --- p.92 / Chapter 3.2.2.1 --- Expression and purification of HIV protease --- p.92 / Chapter 3.2.2.2. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.94 / Chapter 3.2.3 --- Characterization of HIV protease --- p.95 / Chapter 3.2.3.1 --- HIV protease assay by fluorometric measurement --- p.95 / Chapter 3.2.3.2 --- HIV protease assay by using reverse phase high performance liquid chromatography --- p.96 / Chapter 3.3 --- Results --- p.98 / Chapter 3.3.1 --- Expression of HIV protease --- p.98 / Chapter 3.3.2 --- HIV protease assay --- p.98 / Chapter 3.3.2.1 --- Protease assay by using reverse phase HPLC --- p.98 / Chapter 3.3.2.2 --- Protease assay by fluorometric measurement --- p.98 / Chapter 3.3.3 --- Screening of crude Chinese medicinal extracts on inhibition of HIV protease --- p.104 / Chapter 3.3.3.1 --- Methanol extracts --- p.104 / Chapter 3.3.3.2 --- Water extracts --- p.105 / Chapter 3.3.4 --- Characterization of herbal extracts on inhibition of HIV protease --- p.110 / Chapter 3.3.4.1 --- Dose response curve of methanol extract of Woodwardia unigemmata --- p.110 / Chapter 3.3.4.2 --- Dose response curve of hot water extract of Prunella vulgaris --- p.110 / Chapter 3.3.4.3 --- Inhibition mode of methanol extract of Woodwardia unigemmata --- p.113 / Chapter 3.3.4.4 --- Inhibition mode of hot water extract of Prunella vulgaris --- p.113 / Chapter 3.3.4.5 --- Effect of partially purified extracts on HIV protease inhibition --- p.116 / Chapter 3.4 --- Discussion --- p.119 / Chapter Chapter 4 --- General discussion --- p.124 / References --- p.127 / Appendix / Appendix 1 Pictures of herbs used in this study --- p.i / Appendix 2 Mass spectrometry of purified Prunella vulgaris extract --- p.vi / Appendix 3 Calibration curve for determination of HIV PR concentration --- p.viii

Hung, Sze Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 186-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

<p>Rod Salix pripada porodici Salicaceae i obuhvata oko 450 vrsta &scaron;irom sveta, od kojih u Republici Srbiji raste oko 18. Kora vrbe ispoljava antiinflamatorno, antireumatsko, analgetičko i antipiretičko delovanje sinergističkim efektom njenih glavnih aktivnih supstanci &ndash; glikozida salicina, fenolnih i flavonoidnih jedinjenja. Ciljevi ovog rada bili su ispitivanje uticaja klasične i savremenih metoda ekstrakcije na hemijski sastav i biolo&scaron;ke aktivnosti ekstrakata različitih vrsta vrba, utvrđivanje razlika u sadržaju aktivnih komponenti i biolo&scaron;kom potencijalu između ekstrakata kore i ekstrakata lista iste vrste vrbe, kao i utvrđivanje razlika u sadržaju sekundarnih metabolita i antioksidantnoj aktivnosti kod ekstrakata različitih vrsta vrba. Ispitivanja su uključivala analizu 92 ekstrakta kore i lista osam predstavnika roda Salix, dobijenih različitim metodama (maceracija, ultrazvučna i mikrotalasna ekstrakcija) i pri različitim uslovima ekstrakcije (rastvarač, vreme, veličina čestica). Sadržaj ukupnih fenola i flavonoida, kao i antioksidativna aktivnost određeni su spektrofotometrijski. Hemijska karakterizacija ekstrakata vr&scaron;ena je primenom visokoefikasne tečne hromatografije (HPLC). Metodom in silico molekularnog dokinga ispitan je inhibitorni potencijal sastojaka ekstrakata prema enzimima ciklooksigenaze (COX-1 i COX-2) i acetilholinesterazi (AChE). Savremenim metodama ekstrakcije izolovan je veći broj pojedinačnih komponenata u najvećoj koncentraciji i dobijeni su ekstrakti sa jačim potencijalom neutralizacije hidroksilnih radikala. Klasična metoda maceracije 70% etanolom (v/v) bila je pogodnija za dobijanje ekstrakata sa jačim antioksidativnim potencijalom prema DPPH radikalu kod gotovo svih ispitivanih Salix vrsta. Kod većine Salix vrsta jači antioksidantni potencijal prema DPPH radikalu ispoljili su ekstrakti kore. Kod polovine ispitivanih vrsta ekstrakti lista su inhibisali hidroksilne radikale u većoj meri. Veći sadržaj pojedinačnih jedinjenja u ekstraktima kore ili lista zavisio je od same vrste vrbe. Najjaču antioksidantu aktivnost ispoljila je vrsta S. alba, dok je u pogledu hemijskog sastava najraznovrsnija vrsta bila S. fragilis. In silico analizom metodom molekularnog dokinga utvrđen je jak inhibitorni potencijal flavonoidnih jedinjenja kvercetina, naringenina i epikatehina, kao i hlorogenske kiseline među fenolnim kiselinama prema enzimima COX-1 i COX-2. Epikatehin, salicin i hlorogenska kiselina ispoljili su značajno inhibitorno delovanje na enzim AChE u doking studijama. Dobijeni rezultati ukazuju da pored kore i list vrbe ima veliki potencijal primene kao izvor značajnih fitokomponenata, kao i na mogućnost eksploatacije vrsta vrba koje nisu u komercijalnoj upotrebi kao lekovitih sirovina za izolovanje antioksidanasa i farmakolo&scaron;ki aktivnih supstanci.</p> / <p>The genus Salix belongs to the family Salicaceae and comprises about 450 species worldwide, out of which 18 grow in the Republic of Serbia. Willow bark exhibits anti-inflammatory, anti-rheumatic, analgesic and antipyretic effects through synergistic action of its main ingredients -glycosidesalicin, phenolic and flavonoid compounds. Aims of this paper were to analyse the impact of classical and modern extraction methods on chemical composition and biological activities of extracts of different willow species, determine the differences in the amount of active compounds and biological potential between bark and leaf extracts within the same willow species, as well as between extracts of various species. The analysis included 92 bark and leaf extracts of eight species from the genus Salix, obtained by different extraction methods (maceration, ulrasound and microwave assisted extraction) and conditions (solvent, time, particle size). Total phenolics and flavonoids content, as well as antioxidant activity were determined spectrophotometrically. Chemical characterization was carried out by high performance liquid chromatography (HPLC). Cyclooxygenase (COX-1 and COX-2) and acetylcholine esterase (AChE) inhibitory potential of compounds isolated from the extracts was examined by in silico method of molecular docking. Greater number of individual components in highest concentration, as well as exracts with stronger hydroxyl radical scavenging potential were obtained by modern extraction methods. Classical method of maceration with 70% ethanol (v/v) was more suitable for obtaining extracts with higher DPPH radical scavenging activity in the vast majority of tested species. In half of the analysed species leaf extracts inhibited hydroxyl radicals more than bark. Higher amount of individual compounds in bark or leaf extracts depended on the species itself. S. alba exhibited the strongest antioxidant activity, whereas S. fragilis had the most diverse chemical composition. Strong COX-1 and COX-2 inhibitory potential of flavonoids quercetin, naringenin and epicatechin, as well as chlorogenic acid among phenolic acids was determined by in silico molecular docking analysis. Molecular docking studies also demonstrated the inhibitory activity of epicatechin, salicin and chlorogenic acid toward AChE. The obtained results indicate that not only bark, but willow leaf as well could be used as source of significant phytochemicals and also the possibility of exploitation of willow species that are not commercially used as medicinal raw material for isolation of antioxidants and pharmacologycally active substances.</p>

Li Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 146-161). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.III / TABLE OF CONTENTS --- p.VII / LIST OF FIGURES AND TABLES --- p.XIII / LIST OF ABBREVIATIONS --- p.XX / Chapter CHAPTER ONE --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Authentication of Chinese medicines --- p.1 / Chapter 1.1.1 --- The need for authentication of Chinese medicines --- p.1 / Chapter 1.1.2 --- Traditional methods for authentication --- p.2 / Chapter 1.1.3 --- Molecular methods for authentication --- p.4 / Chapter 1.1.3.1 --- DNA fingerprinting --- p.5 / Chapter 1.1.3.2 --- DNA sequencing --- p.6 / Chapter 1.1.3.2.1 --- Choosing a suitable region for DNA sequencing --- p.7 / Chapter 1.1.3.2.2 --- Chloroplast irnL-trnF region --- p.9 / Chapter 1.1.3.2.3 --- Complete sequence of ITS rDNA region --- p.10 / Chapter 1.1.3.2.4 --- 5S rDNA intergenic spacer --- p.11 / Chapter 1.1.3.2.5 --- Calculation of similarities among sequences --- p.12 / Chapter 1.1.3.2.6 --- Construction methods of phylograms --- p.12 / Chapter 1.2 --- The need for molecular authentication of three medicinal herbs --- p.14 / Chapter 1.2.1 --- The herb Baiying --- p.14 / Chapter 1.2.1.1 --- The poisoning case reported in Hong Kong --- p.14 / Chapter 1.2.1.2 --- The identity of genuine Baiying --- p.15 / Chapter 1.2.1.3 --- Morphological characters of the herb Baiying --- p.15 / Chapter 1.2.1.4 --- Medicinal values of Baiying --- p.17 / Chapter 1.2.1.5 --- Xungufeng as the adulterant of Baiying --- p.17 / Chapter 1.2.1.5.1 --- The toxic chemicals aristolochic acids --- p.18 / Chapter 1.2.1.6 --- The need for molecular authentication of Baiying --- p.19 / Chapter 1.2.2 --- The herb Baihuasheshecao --- p.19 / Chapter 1.2.2.1 --- The identity of Baihuasheshecao --- p.19 / Chapter 1.2.2.2 --- Morphological characters of the herb Baihuasheshecao --- p.20 / Chapter 1.2.2.3 --- Medicinal uses --- p.23 / Chapter 1.2.2.4 --- Chemical profile --- p.24 / Chapter 1.2.2.5 --- Adulterants of Baihuasheshecao --- p.24 / Chapter 1.2.2.6 --- Chemical studies of H. diffusa and H. corymbosa --- p.25 / Chapter 1.2.2.7 --- Existing methods for authentication --- p.26 / Chapter 1.2.2.8 --- The need for molecular authentication of Baihuasheshecao --- p.28 / Chapter 1.2.3 --- The herb Chuanlianzi --- p.28 / Chapter 1.2.3.1 --- The identity of Chuanlianzi --- p.28 / Chapter 1.2.3.2 --- Medicinal values --- p.29 / Chapter 1.2.3.3. --- The bioactive chemical --- p.31 / Chapter 1.2.3.4 --- Kulianzi as the substitute of Chuanlianzi --- p.31 / Chapter 1.2.3.5 --- Poisoning cases reported due to ingestion of Kulianzi --- p.32 / Chapter 1.2.3.6 --- Comparative studies of Chuanlianzi and Kulianzi --- p.32 / Chapter 1.2.3.7 --- The need for molecular authentication of Chuanlianzi --- p.33 / Chapter CHAPTER TWO --- OBJECTIVE --- p.35 / Chapter CHAPTER THREE --- MATERIALS AND METHODS --- p.36 / Chapter 3.1 --- Plant and herb samples --- p.36 / Chapter 3.2 --- Total DNA extraction --- p.48 / Chapter 3.2.1 --- Cetyltriethylammonium bromide extraction --- p.48 / Chapter 3.2.2 --- Commercial kit extraction --- p.49 / Chapter 3.3 --- DNA amplification --- p.50 / Chapter 3.4 --- DNA fingerprinting --- p.51 / Chapter 3.4.1 --- DNA concentration determination --- p.51 / Chapter 3.4.2 --- ISSR fingerprinting --- p.52 / Chapter 3.5 --- Agarose gel electrophoresis --- p.53 / Chapter 3.6 --- Purification of PCR product --- p.53 / Chapter 3.7 --- Cloning of PCR product --- p.54 / Chapter 3.7.1 --- Ligation --- p.54 / Chapter 3.7.2 --- Transformation --- p.55 / Chapter 3.7.3 --- Cell cultivation --- p.55 / Chapter 3.7.4 --- Plasmid extraction --- p.55 / Chapter 3.7.5 --- Insert confirmation --- p.56 / Chapter 3.8 --- Determination of DNA concentration --- p.56 / Chapter 3.9 --- DNA sequencing --- p.57 / Chapter 3.9.1 --- Cycle sequencing --- p.57 / Chapter 3.9.2 --- Purification of cycle sequencing products --- p.57 / Chapter 3.9.3 --- DNA analysis --- p.58 / Chapter 3.10 --- Sequence analysis --- p.58 / Chapter CHAPTER FOUR --- MOLECULAR AUTHENTICATION OF BAIYING --- p.59 / Chapter 4.1 --- Results --- p.59 / Chapter 4.1.1 --- Sequence alignment --- p.59 / Chapter 4.1.2 --- Percentage similarity analysis --- p.68 / Chapter 4.1.3 --- Phylogram study --- p.71 / Chapter 4.2 --- Discussion --- p.79 / Chapter 4.2.1 --- Evaluation of chloroplast trnL-trnF region in differentiation of Baiying and Xungufeng --- p.79 / Chapter 4.2.2 --- Molecular authentication of Baiying --- p.80 / Chapter 4.3 --- Conclusion --- p.81 / Chapter CHAPTER FIVE --- MOLECULAR AUTHENTICATION OF BAIHUASHESHECAO --- p.82 / Chapter 5.1 --- Results --- p.82 / Chapter 5.1.1 --- ITS region used for DNA sequencing --- p.82 / Chapter 5.1.2 --- Sequence alignment --- p.82 / Chapter 5.1.3 --- Percentage similarity analysis --- p.88 / Chapter 5.1.4 --- Phylogram study --- p.90 / Chapter 5.2 --- Discussion --- p.98 / Chapter 5.2.1 --- Evaluation of complete sequence of ITS region in differentiation of Hedyotis species --- p.98 / Chapter 5.2.2 --- Molecular authentication of retailed Baihuasheshecao --- p.99 / Chapter 5.2.3 --- Analysis of conflicting data between this study and published results --- p.99 / Chapter 5.2.3.1 --- Comparison of ITS-1 region --- p.101 / Chapter 5.2.3.2 --- Comparison of ITS-2 region --- p.104 / Chapter 5.2.3.3 --- Proposed reasons for the conflicting data --- p.108 / Chapter 5.3 --- Conclusion --- p.109 / Chapter CHAPTER SIX --- MOLECULAR AUTHENTICATION OF CHUANLIANZI --- p.110 / Chapter 6.1 --- Results --- p.110 / Chapter 6.1.1 --- DNA sequencing --- p.110 / Chapter 6.1.1.1 --- Complete sequence of ITS region used for DNA sequencing --- p.110 / Chapter 6.1.1.1.1 --- Sequence alignment --- p.111 / Chapter 6.1.1.1.2 --- Percentage similarity analysis --- p.113 / Chapter 6.1.1.2 --- 5S rDNA intergenic spacer used for DNA sequencing --- p.113 / Chapter 6.1.1.2.1 --- Sequencing alignment --- p.114 / Chapter 6.1.1.2.2 --- Percentage similarity analysis --- p.122 / Chapter 6.1.1.2.3 --- Phylogram study --- p.128 / Chapter 6.1.2 --- ISSR fingerprinting --- p.136 / Chapter 6.2 --- Discussion --- p.138 / Chapter 6.2.1 --- DNA sequencing results --- p.138 / Chapter 6.2.2. --- ISSR fingerprinting results --- p.139 / Chapter 6.2.3 --- Investigation of the identity of retailed Chuanlianzi --- p.140 / Chapter 6.2.4 --- Taxonomic interpretation for Melia species --- p.141 / Chapter 6.2.5 --- Kulianzi involved in this study --- p.141 / Chapter 6.3 --- Conclusion --- p.141 / Chapter CHAPTER SEVEN --- CONCLUSION --- p.143 / BILBIOGRAPHY --- p.146 / APPDENDIX - MATERIAL PREPARATION --- p.162

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Pepper-rosmarin (Lippia sidoides Cham.) is a medicinal and aromatic plant, native Northeast Region of Brazil, which has an essential oil widely used for its properties as bactericide, fungicide, molluscicide, and larvicide. This paper aimed to characterize and evaluate the performance of accessions of pepper-rosmarin (Lippia sidoides Cham.) native to various States of the Northeast Region, kept in Germplasm Bank at São Cristóvão, Sergipe, Brazil. The first chapter presents the general introduction, the literature review on genetic resources and sustainability of agroecosystems, origin, ethnobotany, and germplasm characterization (morphological, agronomic and chemical). The second chapter presents and discusses the data on the morphological and agronomic characterization of accessions of pepper-rosmarin cultivated in field with seedlings from stem cuttings, using a randomized complete block design with two replications. The evaluated variables were: plant height, fresh and dry matter from leaves and stem, width and length of leaves, color of leaves and stem, crown shape, and content and yield of essential oil. Results showed morphological differences among accessions for color of stem, crown shape, plant height, width and length of leaves and ratio length/width of leaves. For agronomic variables, there were significant differences for content and yield of essential oil with the best and worst performance for accessions LSID-005 and LSID-105, respectively. Third chapter presents the data on performance evaluation of pepper-rosmarin, during two years of field cultivation, for the variables: plant height, fresh and dry matter of stem and leaves, content and yield of essential oil, and the content of chemical components of the essential oil. The higher averages found for content (7,68 %) and yield (56,46 mL plant- 1) essential oil were produced by the accession LSID-105, in 2005, and by LSID-102, in 2006, respectively. The higher averages for content of thymol (90,82 %) and carvacrol (56,05 %) were obtained from the accessions LSID-003 and LSID-104, respectively, both in 2006. The accession LSID-104, native to Sergipe State, presented carvacrol as major chemical component. All the other accessions presented thymol as major chemical component. All the accessions, in both years, presented phenotypical variability for all the evaluated variables, except for content of terpil-4-ol. Fourth chapter presents the evaluation of influence of dry and rainy seasons on the performance of accessions of pepper-rosmarin, using the variables: plant height, dry matter of leaves, content and yield of essential oil and content of chemical components of the essential oil. For the majority of the studied accessions, the thymol content was higher during the rainy season. LSID-102 accession presented high yield of essential oil (56,46 mL plant- 1) and thymol content between 47,97 % and 64,95 %. LSID-003 accession yielded 10,06 mL plant-1 of essential oil amd thymol content of 90,02 %. LSID-104 accession presented high content of carvacrol (54,68%) and low content of thymol (7,88 %). Fifth chapter presents the evaluation of the influence of leaves dehydration (at 40 °C for 5 days) on the content of essential oil and its chemical components of 11 accessions of pepper-rosmarin harvested during dry and rainy season. Generally, drying of leaves modified the content of most of the chemical constituents. In relation of the major iv chemical constituents, drying modified thymol content, but did not modified the carvacrol content of the essential oil of the pepper-rosmarin accessions. / Alecrim-pimenta (Líppia sidoides Cham.) é uma planta medicinal e aromática, nativa do Nordeste do Brasil, cujo óleo essencial é utilizado em virtude de suas propriedades bactericida, fungicida, moluscida e larvicida. Este trabalho visou caracterizar e avaliar o comportamento de acessos de alecrim-pimenta (Lippia sidoides Cham.), de vários Estados do Nordeste do Brasil, mantidos em Banco Ativo de Germoplasma em São Cristóvão. No primeiro capítulo, apresentou-se a introdução geral do trabalho, o referencial teórico sobre recursos genéticos e sustentabilidade de agroecossistemas, a origem, os aspectos botânicos e de uso de alecrim-pimenta, a conservação de recursos genéticos e a caracterização de germoplasma (caracterização morfológica, agronômica e química). O segundo capítulo consistiu da caracterização morfológica e agronômica de acessos de alecrim-pimenta cultivados em campo, utilizando-se delineamento experimental em blocos ao acaso com duas repetições. Foram avaliadas as variáveis: altura de planta, massa fresca e seca de folha e caule, largura e comprimento de folha, cor de folha, formato de copa, teor e rendimento de óleo essencial. Os resultados mostraram diferenças morfológicas nos acessos para as variáveis: cor de caule, formato de copa, altura de planta, comprimento e largura de folha e relação comprimento/largura de folha. Para as características agronômicas, foram evidenciadas diferenças significativas para teor e rendimento óleo essencial, sendo o acesso LSID-005 o mais promissor e o acesso LSID-105 o menos promissor. No terceiro capítulo estudou-se o comportamento de acessos de alecrim-pimenta em dois anos de cultivo, e foram avaliadas as variáveis: altura de planta, massa fresca de caule e folha, teor, rendimento e teores dos constituintes químicos do óleo essencial. As maiores médias encontradas para os caracteres teor e rendimento de óleo essencial foram do acesso LSID-105, com 7,68 % no ano de 2005, e do acesso LSID-102, com 56,46 mL planta-1 no ano de 2006, respectivamente. As maiores médias de timol e de carvacrol foram encontradas no acesso LSID-003, com 90,82 % e do acesso LSID-104, com 56,05 % respectivamente, ambos no ano de 2006. O acesso LSID-104, oriundo do Estado de Sergipe, apresentou como constituinte químico majoritário o carvacrol. Os demais acessos têm o timol como constituinte químico majoritário. Todos os acessos, nos dois anos de cultivo, apresentaram variabilidade fenotípica para todas as variáveis analisadas, exceto para teor de terpil 4-ol. No quarto capítulo, avaliou-se a influência das épocas seca e chuvosa no comportamento de acessos de alecrim-pimenta, avaliandose as variáveis: massa fresca e seca de folha, teor e rendimento do óleo essencial e os teores dos constituintes químicos do óleo essencial. Observou-se que, para a maioria dos acessos de alecrim-pimenta estudados, o teor de timol é maior na época chuvosa do que na seca. O acesso LSID-102 apresentou alto rendimento de óleo essencial, porém com teor de timol entre 47,97 % e 64,95 %. O acesso LSID-003 apresentou rendimento de óleo essencial de 10,06 mL planta-1 e teor de timol de 90,02 % e, o acesso LSID-104 apresentou alto teor de carvacrol (média de 54,68 %) e baixo teor de timol (média de 7,88 %). No quinto capítulo, estudou-se a influência da secagem das folhas no óleo essencial de acessos de alecrim-pimenta. Testou-se a influência da secagem das folhas a 40º C por cinco dias em 11 acessos de alecrim-pimenta, colhidas nas estações seca e chuvosa. Avaliaram-se o teor de óleo essencial e os teores dos constituintes químicos no ii óleo essencial. A secagem proporcionou redução de teor de óleo essencial de alecrimpimenta quando colhido na estação seca. Em geral, a secagem alterou os teores da maioria dos constituintes químicos. Em relação aos constituintes químicos majoritários, a secagem alterou o teor de timol, mas não alterou o teor de carvacrol do óleo essencial de acessos de alecrim-pimenta.