No description available.

The cells of the licorice plant, Glycyrrhiza glabra, were cultured as a "single cell" suspension. Their growth behaviour, yield and metabolic products were studied. The suspension cultures of the licorice plant were established from the friable calluses obtained from the radicle, cotyledon and hypocotyl of the germinated seeds. The single cells, regardless of their origin showed little difference in cell size and morphology. After an apparent adjustment to the medium, the cells required 11-13 days of incubation to reach the maximum cell yield of 1.2 gm/100 ml medium, dry weight. During the growth period, the pH of the growth medium decreased from pH 5.6 to pH 4.7 in the first few days and then increased to about pH 6. A level of 10% coconut milk in PRL-4-CM medium was found to support good cell growth; the lower the coconut milk level, the longer the growth period required to reach the maximum cell yield. It was also found that 0.57% yeast extract could be used to replace the coconut milk in the PRL-4-CCM medium. The metabolites detected and examined in the licorice single cell suspension culture included a volatile apple aroma, a polysaccharide pectin-like material, steroids and triterpenoids. The analyses of the licorice cell volatile apple aroma found under anaerobic conditions indicated the presence of ethanol and some related esters. The monosaccharides found in the pectin-like polysaccharide hydrolysate were glucose, fructose, galactose, arabinose, xylose, galacturonic acid and glucuronic acid. The pectin-like material in the cell preparations reached a maximum yield of 1.1 mg/ml after one month of growth. Glycyrrhizinic acid, the common licorice constituent found in the root, could not be detected in the suspension cultures. However, several other related compounds which gave typical steroid and triterpenoid reactions were found. Sorbitol and fructose were found to be the two major sugars which accumulated in free form in the licorice cell medium. / Land and Food Systems, Faculty of / Graduate

Lee Kin-wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 187-203). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vi / CHINESE ABSTRACT --- p.ix / TABLE OF CONTENTS --- p.xii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Neuroblastoma-An Overview --- p.1 / Chapter 1.1.1 --- Epidemiology of Neuroblastoma --- p.1 / Chapter 1.1.2 --- Clinical Presentations of Neuroblastoma --- p.2 / Chapter 1.1.3 --- Diagnosis of Neuroblastoma --- p.3 / Chapter 1.1.4 --- Staging of Neuroblastoma --- p.5 / Chapter 1.1.5 --- Prognostic Considerations of Neuroblastoma --- p.6 / Chapter 1.1.5.1 --- Age --- p.6 / Chapter 1.1.5.2 --- Primary Tumor Site --- p.6 / Chapter 1.1.5.3 --- Tumor Histopathology --- p.7 / Chapter 1.1.5.4 --- Serum Markers --- p.9 / Chapter 1.1.5.5 --- Genetic Markers --- p.9 / Chapter 1.1.6 --- Standard Treatment Modalities for Neuroblastoma --- p.11 / Chapter 1.1.6.1 --- Surgery --- p.11 / Chapter 1.1.6.2 --- Chemotherapy --- p.12 / Chapter 1.1.6.3 --- Radiotherapy --- p.13 / Chapter 1.1.7 --- Differentiation of Neuroblastoma In Vivo and In Vitro --- p.14 / Chapter 1.1.8 --- Differentiation Therapy of Neuroblastoma --- p.16 / Chapter 1.2 --- Glycyrrhizin ´ؤ the Major Active Component of Licorice --- p.17 / Chapter 1.2.1 --- Chemistry of Licorice --- p.17 / Chapter 1.2.2 --- Metabolism of Glycyrrhizin --- p.21 / Chapter 1.2.3 --- Pharmacological Effects of Glycyrrhizin and 18β-Glycyrrhetinic Acid --- p.22 / Chapter 1.2.3.1 --- Anti-inflammatory Effect --- p.22 / Chapter 1.2.3.2 --- Hepatoprotective Effect --- p.23 / Chapter 1.2.3.3 --- Anti-carcinogenic and Anti-tumor Effects --- p.25 / Chapter 1.2.3.4 --- Anti-viral Effect --- p.27 / Chapter 1.2.3.5 --- Immunomodulatory Effect --- p.29 / Chapter 1.2.3.6 --- Mineralocorticoid Effect --- p.30 / Chapter 1.2.4 --- Pharmacokinetics of Glycyrrhizin and Glycyrrhetinic Acid --- p.32 / Chapter 1.2.5 --- Health Hazards of Glycyrrhizin and Glycyrrhetinic Acid --- p.33 / Chapter 1.3 --- Aims and Scopes of This Study --- p.36 / Chapter CHAPTER 2: --- MATERIALS AND METHODS --- p.39 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Cell Lines --- p.39 / Chapter 2.1.2 --- "Cell Culture Media, Buffers and Other Reagents" --- p.39 / Chapter 2.1.3 --- Drugs and Chemicals --- p.43 / Chapter 2.1.4 --- Reagents for 3H-Thymidine Incorporation Assay --- p.44 / Chapter 2.1.5 --- Reagents for Neutral Red Assay --- p.45 / Chapter 2.1.6 --- Reagents for Clonogenic Assay --- p.45 / Chapter 2.1.7 --- Reagents and Buffers for Immunocytochemistry --- p.46 / Chapter 2.1.8 --- Reagents for DNA Extraction --- p.48 / Chapter 2.1.9 --- Reagent for DNA Staining --- p.49 / Chapter 2.1.10 --- Reagents and Buffers for Flow Cytometry --- p.49 / Chapter 2.1.11 --- Reagents for Total RNA Isolation --- p.50 / Chapter 2.1.12 --- Reagents and Buffers for RT-PCR --- p.50 / Chapter 2.1.13 --- Reagents and Buffers for Gel Electrophoresis --- p.55 / Chapter 2.1.14 --- Reagents and Buffers for Western Blot Analysis --- p.56 / Chapter 2.2 --- Methods --- p.62 / Chapter 2.2.1 --- Cell Culture Methodology --- p.62 / Chapter 2.2.2 --- Determination of Cell Proliferation --- p.62 / Chapter 2.2.3 --- Determination of Cell Viability by Trypan Blue Exclusion Test --- p.64 / Chapter 2.2.4 --- Limiting Dilution Assay --- p.65 / Chapter 2.2.5 --- Clonogenic Assay --- p.65 / Chapter 2.2.6 --- Measurement of Apoptosis by DNA Fragmentation Analysis --- p.66 / Chapter 2.2.7 --- Assessment of Apoptosis by Hoechst 33342 Staining --- p.67 / Chapter 2.2.8 --- Cell Morphological Study --- p.67 / Chapter 2.2.9 --- Immunocytochemistry --- p.68 / Chapter 2.2.10 --- Flow Cytometric Analysis of Cell Cycle Profile --- p.69 / Chapter 2.2.11 --- Gene Expression Study --- p.70 / Chapter 2.2.12 --- Protein Expression Study --- p.73 / Chapter 2.2.13 --- Statistical Analysis --- p.76 / Chapter CHAPTER 3: --- ANTI-PROLIFERATIVE EFFECTS OF GLYCYRRHIZIN AND 18β-GLYCYRRHETINIC ACID ON NEUROBLASTOMA CELLS --- p.77 / Chapter 3.1 --- Introduction --- p.77 / Chapter 3.2 --- Results --- p.79 / Chapter 3.2.1 --- Differential Anti-proliferative Effect of Glycyrrhizin and 18β- Glycyrrhetinic Acid on Various Neuroblastoma Cell Lines In Vitro --- p.79 / Chapter 3.2.2 --- Effect of 18P-Glycyrrhetinic Acid on the Clonogenicity of the Murine Neuroblastoma BU-1 Cells In Vitro --- p.91 / Chapter 3.2.3 --- Kinetic and Reversibility Studies of the Anti-proliferative Effect of Glycyrrhizin and 18β-Glycyrrhetinic Acid on the Neuroblastoma BU-1 Cells --- p.93 / Chapter 3.2.4 --- Cytotoxic Effect of Glycyrrhizin and 18β-Glycyrrhetinic Acid on the Neuroblastoma BU-1 Cells In Vitro --- p.100 / Chapter 3.2.5 --- Inability of Glycyrrhizin and 18β-Glycyrrhetinic Acid to Induce DNA Fragmentation in the Neuroblastoma BU-1 Cells --- p.102 / Chapter 3.3 --- Discussion --- p.107 / Chapter CHAPTER 4: --- DIFFERENTIATION-INDUCING EFFECTS OF GLYCYRRHIZIN AND 18β-GLYCYRRHETINIC ACID ON NEUROBLASTOMA CELLS --- p.112 / Chapter 4.1 --- Introduction --- p.112 / Chapter 4.2 --- Results --- p.114 / Chapter 4.2.1 --- Morphological Changes in Glycyrrhizin and 18β-Glycyrrhetinic Acid-treated Neuroblastoma BU-1 Cells --- p.114 / Chapter 4.2.2 --- Immunocytochemistry of Glycyrrhizin and 18β-Glycyrrhetinic Acid-treated Neuroblastoma BU-1 Cells --- p.118 / Chapter 4.2.3 --- Effect of 18β-Glycyrrhetinic Acid on the Expression of Proto-oncogenes in Neuroblastoma BU-1 Cells --- p.124 / Chapter 4.2.4 --- Effect of 18β-Glycyrrhetinic Acid on the Expression of Differentiation-Related Genes in Neuroblastoma BU-1 Cells --- p.127 / Chapter 4.3 --- Discussion --- p.130 / Chapter CHAPTER 5: --- MECHANISTIC STUDIES ON THE ANTI-PROLIFERATIVE AND DIFFERENTIATION-INDUCING EFFECTS OF GLYCYRRHIZIN AND 18β-GLYCYRRHETINIC ACID --- p.136 / Chapter 5.1 --- Introduction --- p.136 / Chapter 5.2 --- Results --- p.139 / Chapter 5.2.1 --- Effects of Glycyrrhizin and 18β-Glycyrrhetinic Acid on the Cell Cycle Kinetics of Neuroblastoma BU-1 Cells In Vitro --- p.139 / Chapter 5.2.2 --- Modulatory Effects of 18β-Glycyrrhetinic Acid on the Expression of Cell Cycle Regulatory Genes and Proteins --- p.145 / Chapter 5.2.3 --- "Combined Effects of Glycyrrhizin, 18β-Glycyrrhetinic Acid and All-Trans Retinoic Acid on the Proliferation of Neuroblastoma BU-1 Cells In Vitro" --- p.149 / Chapter 5.2.4 --- "Combined Effects of Glycyrrhizin, 18β-Glycyrrhetinic Acid and All-Trans Retinoic Acid on the Differentiation of Neuroblastoma BU-1 Cells In Vitro" --- p.153 / Chapter 5.2.5 --- Modulatory Effect of 18β-Glycyrrhetinic Acid on the Expression of PKC Isoforms in Neuroblastoma BU-1 Cells --- p.156 / Chapter 5.2.6 --- The Possible Involvement of Protein Kinase C in the Anti-proliferative and Differentiation-Inducing Effects of 18β-Glycyrrhetinic Acid on the Neuroblastoma BU-1 Cells --- p.158 / Chapter 5.2.7 --- The Possible Involvement of Protein Kinase A in the Anti-proliferative and Differentiation-Inducing Effects of 18β-Glycyrrhetinic Acid on the Neuroblastoma BU-1 Cells --- p.165 / Chapter 5.3 --- Discussion --- p.173 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.182 / REFERENCES --- p.187

Leung Yuet Kin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 147-162). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 2004. / Also available in print.

肺癌是導致男性死亡的最常見原因以及是排在乳腺癌和結腸癌之後的導致女性死亡的第三大原因。雖然肺癌如此嚴重，但是如今治疗肺癌仍然是一个挑战。現今對肺癌的治療主要集中在化學治療和靶點藥物治療，但是由於這些治療有著很大的副作用和低治愈率，尋找其他的醫學替代方法十分迫切。甘草是其中最常用的中藥，它常常用作食品工業中的甜味劑。以往的研究表明，甘草具有多種的生物活性。但是甘草提取物對於肺癌的治療卻是十分匱乏的。 / 本論文主要目的是評價甘草提取物以及其中的有效成份對非小型肺癌細胞株A549 的影響，以及其作用的機理。我們的數據表明，甘草的乙酸乙酯（EAL）成份比甘草的乙醇提取物有著比較強的抑制癌細胞的作用。另外，對甘草的五個單體進行的測試中發現lico-3 是最具有抑制肺癌作用的。利用高效液相色譜法對甘草活性成份分析表明，lico-3 是EAL中的其中一個單體。 / 乳酸脫氫酶滲漏（LDH）的檢測結果以及异硫氰酸荧光素-碘化丙啶（FITC-PI）雙染的結果表明，EAL 能夠引起肺癌細胞的凋亡現象而非壞死現象。實驗結果表明由EAL引起的A549細胞凋亡是跟Bcl-2家族及Caspase家族有關係，同時EAL還能夠抑制Akt途徑從而導致細胞的死亡。 / 致肺癌細胞死亡的原因進行進一步研究表明，EAL還能夠引起抑制細胞週期的運作，停留在G2/M 時期。這可能是由於EAL引發了p53與p21的上調作用從而抑制了細胞的生長與增殖。 / 實驗結果說明了EAL引起的肺癌細胞株A549的凋亡作用是跟多重細胞通路有關， 同時表明了EAL是具有抗擊肺癌作用的潛能，能夠作為治療肺癌的藥物。 / Lung cancer is the most common cause of cancer death in men and third in women followed by breast cancer and colon cancer, yet treatment of lung cancer remains a challenge. Current treatments including chemotherapy and targeted drug treatment come with side-effects and low successful rate. Alternative medicine for treatment of lung cancer is warranted. Glycyrrhiza uralensis (Gan-Cao), commonly called “licorice, is one of the most commonly used herbs in traditional Chinese medicine (TCM). It is also used as flavoring and sweetening agents in many of food products. Previous studies have indicated that licorice exhibits a variety of biological activities. However, anticancer effects of licorice extract on lung cancer remain unclear. / In this study, we evaluated effects of licorice extract and its chemical components on human lung cancer cell line A549, and studied its mode of action. Our results showed the ethyl acetate fraction of licorice (EAL) was more effective in inhibition of A549 cell growth followed by ETL (IC₅₀: 50μg/mL). Moreover, among the five compounds tested, lico-3 was more potent compound. The HPLC analysis of the active fraction indicated that lico-3 was one of the compounds distributed in the EA fraction. / The results of LDH assay and FITC-PI co-staining method suggested low concentration of EAL can trigger apoptosis but not necrosis. The experimental findings show that EAL induce apoptosis in A549 cell lines involved in Bcl-2 family and caspase cascade. Also, EAL can arrest the Akt survival pathway in A549. Furthermore, the results indicate that EAL triggered G2/M phase arrest. The studies suggest EAL can up-regulate p53 and p21 to promote cell cycle arrest resulting in inhibition of proliferation. / Experimental results indicate that EAL is involved in multiple signal pathways to induce lung cancer cell death. The result suggests EAL is a potential candidate for lung cancer therapy. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Yanling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 99-110). / Abstracts in Chinese. / Abstract --- p.III / 論文摘要 --- p.V / Acknowledgement --- p.VII / List of Contents --- p.VIII / List of Figures --- p.X / List of Tables --- p.XI / List of Abbreviations --- p.XII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Lung cancer --- p.1 / Chapter 1.1.1 --- Overview --- p.1 / Chapter 1.1.2 --- Risk factors --- p.2 / Chapter 1.1.3 --- Types of lung cancer --- p.4 / Chapter 1.1.4 --- Stages and treatment of lung cancer --- p.5 / Chapter 1.1.5 --- Chemotherapy for lung cancer treatment --- p.8 / Chapter 1.2 --- Traditional Chinese Medicines --- p.11 / Chapter 1.2.1 --- Overview --- p.11 / Chapter 1.2.2 --- Licorice --- p.14 / Chapter 1.2.3 --- Chemical study of licorice --- p.16 / Chapter 1.2.4 --- Pharmacological activities of licorice --- p.16 / Chapter 1.3 --- Molecular mechanism of apoptosis --- p.21 / Chapter 1.3.1 --- Overview --- p.21 / Chapter 1.3.2 --- Bcl2 family --- p.21 / Chapter 1.3.3 --- Caspase pathway --- p.23 / Chapter 1.3.4 --- Akt pathway --- p.24 / Chapter 1.3.5 --- p53 protein --- p.26 / Chapter 1.3.6 --- Apoptosis and cancer --- p.27 / Chapter 1.4 --- Cell cycle --- p.29 / Chapter 1.4.1 --- Overview --- p.29 / Chapter 1.4.2 --- Cell cycle and p53 --- p.29 / Chapter 1.4.3 --- Cell cycle and cancer --- p.30 / Chapter 1.5 --- Aims of study --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- Cell culture and treatment --- p.33 / Chapter 2.1.1 --- Cell line --- p.33 / Chapter 2.1.2 --- Chemicals and reagents --- p.34 / Chapter 2.1.3 --- Preparation of solutions --- p.34 / Chapter 2.2 --- Preparation of Licorice sample --- p.35 / Chapter 2.3 --- HPLC analysis --- p.35 / Chapter 2.3.1 --- Chemical and materials --- p.35 / Chapter 2.3.2 --- Instrumentation --- p.36 / Chapter 2.3.3 --- Preparation of Standard solutions --- p.36 / Chapter 2.3.4 --- Preparation of samples --- p.37 / Chapter 2.3.5 --- HPLC conditions --- p.37 / Chapter 2.3.6 --- Method validation --- p.37 / Chapter 2.4 --- Cell viable assay --- p.38 / Chapter 2.4.1 --- Samples preparation --- p.39 / Chapter 2.4.2 --- Procedure --- p.39 / Chapter 2.5 --- LDH assay --- p.40 / Chapter 2.5.1 --- Reagent preparation --- p.40 / Chapter 2.5.2 --- Procedure --- p.41 / Chapter 2.6 --- Annexin V assay --- p.41 / Chapter 2.6.1 --- Reagent --- p.42 / Chapter 2.6.2 --- Procedure --- p.42 / Chapter 2.7 --- Cell cycle study --- p.43 / Chapter 2.7.1 --- Chemicals and reagent --- p.43 / Chapter 2.7.2 --- Procedure --- p.44 / Chapter 2.8 --- Caspase3/7 Assay --- p.44 / Chapter 2.8.1 --- Reagent preparation --- p.45 / Chapter 2.8.2 --- Procedure --- p.46 / Chapter 2.9 --- Western blotting --- p.46 / Chapter 2.9.1 --- Reagent and antibodies --- p.46 / Chapter 2.9.2 --- Procedure --- p.50 / Chapter 2.9.3 --- Determination of protein concentration --- p.51 / Chapter 2.10 --- Data analysis --- p.51 / Chapter Chapter 3 --- Results --- p.52 / Chapter 3.1 --- Chromatographic conditions and HPLC identity conformation --- p.52 / Chapter 3.1.1 --- Linearity, limits of detection and quantification --- p.56 / Chapter 3.1.2 --- Reproducibility --- p.56 / Chapter 3.1.3 --- Analysis of ethyl acetate of licorice (EAL) using the validated method --- p.56 / Chapter 3.2 --- Licorice induces apoptosis in nonsmall cell lung carcinoma --- p.61 / Chapter 3.2.1 --- Cell viability assay --- p.61 / Chapter 3.2.2 --- LDH leakage assay --- p.71 / Chapter 3.2.3 --- Annexin V and PI staining --- p.73 / Chapter 3.3 --- Protein expression in EALinduced apoptotic cells --- p.75 / Chapter 3.3.1 --- Bcl2 family --- p.75 / Chapter 3.3.2 --- Activation of caspases by EAL treatment --- p.77 / Chapter 3.4 --- EAL could block Akt survival pathway --- p.79 / Chapter 3.5 --- EAL induces cell cycle arrest in nonsmall cell lung carcinoma --- p.83 / Chapter Chapter 4 --- Discussion --- p.85 / Chapter 4.1 --- Chemical analysis of licorice --- p.85 / Chapter 4.2 --- Licorice induced apoptosis but not necrosis on lung cancer cell A549 --- p.86 / Chapter 4.2.1 --- Licorice exhibits specific cytotoxicity to different cancer cells in vitro --- p.86 / Chapter 4.2.2 --- EAL induces cell death via apoptosis but not necrosis --- p.87 / Chapter 4.3 --- Growth inhibition by EAL inducing apoptosis --- p.89 / Chapter 4.3.1 --- EAL induces apoptotic cell death through modification of Bcl2 family --- p.89 / Chapter 4.3.2 --- EAL activate the caspase proteins --- p.90 / Chapter 4.4 --- Growth inhibition by EAL inducing survival pathway arrest --- p.92 / Chapter 4.5 --- Growth inhibition by EAL inducing cellcycle arrest --- p.94 / Chapter 4.6 --- General discussion --- p.96 / Reference --- p.99

Background and purpose: Rhizoma Chuanxiong (CX), the dry rhizome of Ligusticum chuanxiong Hort., is a commonly used Chinese herbal medicine to treat gynecological diseases. So far, more than 60 chemical components have been identified from CX such as volatile oils (ligustilide, etc.), phenolic acids (ferulic acid, etc.) and alkaloids (chuanxiongzine, etc.). These components in CX are the basis of its wide pharmacodynamic actions including estrogen-like, progesterone-like and anti-coagulant/anti- platelet effects. In our recent survey based on previous published clinical trials, CX was ranked as one of the top 20 herbs commonly used for anti-miscarriages amongst Chinese pregnant women. However, CX should be used with caution during pregnancy as its property of 2invigorating blood circulation and removing blood stagnation3. Despite its wide applications, the safe dosage of CX in pregnant women remains unclear with no records found in the current Chinese Pharmacopoeia or other guidelines. Thus, verification regarding the impacts of CX preparations and its components in embryonic development is urgently required. In view of the limited experimental evidence that is currently available to assess the safety of CX, this project aims to (1) identify the general impacts of CX aqueous extract in maternal function and fetal development with an in vivo mouse model; and to (2) investigate the adverse impacts and underlying mechanisms of CX aqueous extract in fetal bone development with a biomarker assay and metabolomics analysis.;Concusion: CX aqueous extract at a low dosage of 2 g/kg/day (equals to the daily dosage of human adults) did not cause adverse effect in pregnant mice, and it suggested that this dosage of CX preparations should be safe for pregnant women. Our data demonstrated that high dosage and long-term use of CX aqueous extract might result in embryonic toxicities including fetal bone malformations for the first time. As the CX aqueous extract in this study was not contaminated by pesticide residues and heavy metals, the adverse impacts of CX aqueous extract should be considered as a result of its intrinsic components in the herb. Furthermore, CX aqueous extract might significantly down-regulate biomarkers related to bone formation and metabolism during osteogenesis. It is therefore valuable to establish a practical approach to systematically assess the safety of CX and other herbal medicines.;Method: Referred to the guidelines of WHO, the Chinese Pharmacopoeia and the Hong Kong Chinese Materia Medica Standards, CX aqueous extract was prepared, and its reference marker (ligustilide and ferulic acid) were quantitatively authenticated by HPLC analysis. LC/MS fingerprint analysis was performed for the quality control purposes. In addition, pesticide residues and heavy metals found in CX aqueous extract were examined using GC-MS and ICP-MS analysis. In the Segment II study as per FDA and OECD guidelines, pregnant mice were randomly assigned into 6 groups (n=18 per group): i.e. mice were orally administrated with distilled water as the negative controls (Group 1); or CX aqueous extract of 2, 16, 24 and 32 g/kg/day respectively from the gestation day (GD)6 to 16(Group 2, 3, 4 and 5); or vitamin A (200,000 IU) on GD7, 9 and 11 as the positive controls (Group 6). All mice were sacrificed to assess maternal and fetal parameters on the GD18. In the mechanistic study, the expressions of biomarkers related to fetal bone development including PICP, ICTP, B-ALP, BGP, Gdf-5, BMPs, BMP-6, BMP-8, BMP-11, IL-4, IL-4r, IL-10 and IL-10r in fetal tissue samples of the Group 1 and 5 (32 g/kg/day, n=18) were measured using ELISA analyses on GD16. Meanwhile, the metabolites of two-group samples were also analyzed by the UHD Accurate-Mass Q- TOF LC/MS, and profiling data was further analyzed by specific software. During statistical analysis, measurement data from G1, 2, 3 4 and 5 groups were analyzed using one-way ANOVA(SPSS software, version 16.0). LSD test in Post hoc method was applied to compare differences between every two groups. Pearsons x 2 - test was used to analyze category data from G1, 2, 3, 4 and 5 groups, and Fishers exact test was applied to compare differences between different groups. The student t-test was also used to compare differences between G1 and G6 groups in animal studies as well as G1 and G5 groups using ELISA or metabolomics results. An intragroup difference with a p-value less than 0.05 was considered as statistical significant.;Resutt:(1) There was no statistical significant difference in maternal and fetal parameters found between the Group 1 and 2 (p> 0.05). However, the maternal body weight (BW), gravid uterine weight, corrected BW change, live fetus/litter, mean fetal BW in the Group 4 and 5 were significantly lower than those in the Group 1(p

Работа состоит из введения, основной части (обзор литературы, материала и методы, результаты и обсуждения), заключения и списка литературы. Объектом исследования были каллусные культуры солодки, а также корневище. Цель работы - подобрать условия культивирования, определить содержание суммы фенолов и флавоноидов и антиоксидантную активность каллусной культуры солодки, в сравнении с интактным корневищем. Были получены асептические клеточные линии для двух генотипов солодки. Проведен подбор оптимальных для наращивания биомассы соотношений фитогормонов - наибольшая доля выживших каллусов получена при сочетании фитогормонов: 0,2 мг/л БАП + 20 мг/л НУК, 1 БАП 2 НУК, 1 БАП 20 НУК. Был проведен анализ содержания суммы фенолов и флавоноидов, а также антиоксидантной активности в этанольных экстрактах каллусов и корневища. Показано, что клеточные культуры имеют перспективное значение в качестве получения фенолов и антиоксидантов. Выпускная квалификационная работа магистра – 51 стр., 13 рис., 52 литературных источников, из которых русскоязычных 37, англоязычных 15. / The work consists of an introduction, the main part (review of literature, material and methods, results and discussions), conclusion and list of references. The object of the study was the licorice callus culture, as well as the rhizome. The purpose of the work is to select cultivation conditions, to determine the content of the sum of phenols and flavonoids and antioxidant activity of licorice callus culture, in comparison with the intact rhizome. Aseptic cell lines were obtained for two licorice genotypes. The ratios of phytohormones optimal for biomass growth were selected - the largest share of surviving calli was obtained with a combination of phytohormones: 0.2 mg / L 6-BA + 20 mg / L NАА, 1 6-BA 2 NАА, 1 6-BA 20 NАА. An analysis was carried out of the content of the sum of phenols and flavonoids, as well as the antioxidant activity in ethanol extracts of callus and rhizome. Cell cultures have been shown to have promising value as phenols and antioxidants. The final qualifying work of the master is 51 pages, 13 figures, 52 literary sources, of which 37 are Russian-speaking and 15 are English-speaking.

Lee Kai Woo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 90-104). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Thesis Committee --- p.iii / Abstracts --- p.v / 論文槪要 --- p.vii / List of figures --- p.viii / List of abbreviations --- p.ix / Chapter Chapter one --- Introduction --- p.1 / Chapter 1.1 --- Drug metabolism and UGTs --- p.1 / Chapter 1.2 --- Natural substrates of UGTs --- p.4 / Chapter 1.3 --- Functions of UGT isoforms: roles of UGT polymorphisms --- p.6 / Chapter 1.4 --- Evolution of the UGT1 gene locus in vertebrates --- p.8 / Chapter 1.5 --- Multiple Variable First Exons: A Mechanism for Cell- and Tissue-Specific Gene regulation --- p.13 / Chapter 1.6 --- Evolutionary Origin of the Variable and Constant Genomic Organization --- p.14 / Chapter 1.7 --- Variable and Constant Genomic Organizations Exist in Mammalian UGTs --- p.20 / Chapter 1.8 --- The history of recombinant UGT expression --- p.20 / Chapter 1.9 --- UGT1A8 --- p.21 / Chapter 1.10 --- Licorice and its active component --- p.24 / Chapter 1.11 --- Enzyme induction in the liver --- p.25 / Chapter 1 12 --- Objectives --- p.28 / Chapter Chapter two --- Methods and Materials --- p.29 / Chapter 2.1 --- UGT1A8 induction studies --- p.30 / Chapter 2.1.1 --- Drug preparation --- p.30 / Chapter 2.1.2 --- Cell viability study with Neutral Red Assay Rat treatment --- p.30 / Chapter 2.1.3 --- Cell treatment --- p.31 / Chapter 2.1.4 --- Rat treatment --- p.31 / Chapter 2.1.5 --- RNA extraction from rat liver and cell culture --- p.31 / Chapter 2.1.6 --- Quantization of RNA --- p.32 / Chapter 2.1.7 --- Denaturing gel electrophoresis for RNA --- p.33 / Chapter 2.1.8 --- Northern hybridization --- p.33 / Chapter 2.1.9 --- Probe for Northern Blotting --- p.34 / Chapter 2.1.10 --- Agarose Gel analysis and Northern Blot analysis --- p.34 / Chapter 2.2 --- Recombinant expression of UGT1A8 in E.coli JM109 --- p.35 / Chapter 2.2.1 --- cDNA synthesis --- p.35 / Chapter 2.2.2 --- Polymerase chain reaction --- p.35 / Chapter 2.2.3 --- Agarose gel electrophoresis for DNA --- p.35 / Chapter 2.2.4 --- "Amplification of target gene, UGT1A8" --- p.36 / Chapter 2.2.5 --- Restriction enzyme digestion of plasmid and insert --- p.36 / Chapter 2.2.6 --- Ligation of plasmid and insert DNA --- p.37 / Chapter 2.2.7 --- Amplification of target plasmid --- p.37 / Chapter 2.2.8 --- Screening of target plasmid --- p.37 / Chapter 2.2.9 --- DNA sequencing --- p.38 / Chapter 2.2.10 --- Transformation of protein expression host --- p.38 / Chapter 2.2.11 --- Confirmation of transformation of protein expression host --- p.38 / Chapter 2.2.12 --- Protein expression --- p.39 / Chapter 2.2.13 --- Protein purification --- p.39 / Chapter 2.2.14 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.40 / Chapter 2.2.15 --- Confirmation of the protein --- p.40 / Chapter 2.3 --- Characterization of recombinant UGT1A8 --- p.41 / Chapter 2.3.1 --- UGT assay --- p.41 / Chapter 2.4 --- Routine experiment methods --- p.41 / Chapter 2.4.1 --- Determination of protein --- p.41 / Chapter 2.4.2 --- Nucleic acid purification --- p.42 / Chapter 2.4.3 --- Preparation of chemically competent bacterial cells --- p.42 / Chapter 2.4.4 --- Colony PCR --- p.43 / Chapter 2.4.5 --- Plasmid rescue by alkaline lysis --- p.44 / Chapter 2.4.6 --- Charging of His-tagged column --- p.44 / Chapter 2.4.7 --- Washing of His-tagged column --- p.45 / Chapter Chapter three --- Results --- p.46 / Chapter 3.1 --- UGT1A8 Expression in clone9 and H4IIE after treatment with licorice and 18 β glycyrrhentinic acid --- p.46 / Chapter 3.2 --- UGT1A8 induction in wistar and j/j rats after treatment --- p.63 / Chapter 3.3 --- Construction of pRset-UGT 1A8 Vector --- p.70 / Chapter 3.4 --- Purification of recombinant UGT1A8 --- p.75 / Chapter 3.5 --- Screening of substrate of the purified enzyme --- p.77 / Chapter Chapter four --- Discussion --- p.78 / Chapter 4.1 --- Effects of licorice and 18βglycyrrhetinic acid in the induction of UGT1A8 in different cell lines --- p.78 / Chapter 4.2 --- Comparison of wistar and j/j rats in the induction of UGT1A8 --- p.79 / Chapter 4.3 --- Comparison of licorice and 18(3 glycyrrhetinic acid in the induction of UGT1A8 in rats --- p.81 / Chapter 4.4 --- Comparison of in vivo and in vitro of drug treatment --- p.81 / Chapter 4.5 --- Expression of UGT1A7 after drug treatment in vitro --- p.82 / Chapter 4.6 --- Protein expression and purification --- p.83 / Chapter 4.7 --- Substrates of UGT1A8 --- p.83 / Chapter Chapter Five --- Conclusions --- p.86 / References --- p.90 / Appendix --- p.105