Yip Nga-lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 151-164). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese Version) --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.6 / Chapter 2.1 --- Medicinal Plants and Herbs --- p.6 / Chapter 2.1.1 --- Antitumor activities --- p.8 / Chapter 2.1.1.1 --- Successful examples - Camptothecin --- p.8 / Chapter 2.1.1.2 --- Successful examples - Taxol --- p.13 / Chapter 2.2 --- Compositae and its Traditional Functions --- p.16 / Chapter 2.3 --- Antitumor Activity of Compositae --- p.19 / Chapter 2.3.1 --- Emilia sonchifolia --- p.19 / Chapter 2.3.2 --- Silymarin --- p.20 / Chapter 2.4 --- Wedelia Species --- p.22 / Chapter 2.4.1 --- Hepatoprotective effect --- p.22 / Chapter 2.4.2 --- Hypoglycemic effect --- p.23 / Chapter 2.4.3 --- Antimicrobial activity --- p.23 / Chapter 2.4.4 --- Antinociceptive activity --- p.24 / Chapter 2.4.5 --- Antifeedant and antifungal activities --- p.24 / Chapter 2.4.6 --- Trypanosomicidal effect --- p.25 / Chapter 2.4.7 --- Chemical constituents --- p.25 / Chapter 2.5 --- Sesquiterpene lactone --- p.27 / Chapter 2.6 --- Cell Cycle Control and Apoptosis --- p.30 / Chapter 2.6.1 --- Cell cycle and its control in cancer therapy --- p.30 / Chapter 2.6.2 --- Apoptosis --- p.36 / Chapter 2.7 --- Four Selected Compositae Species Used in the Study --- p.42 / Chapter 2.7.1 --- Dendranthema indicum (野菊花) --- p.42 / Chapter 2.7.2 --- Dendranthema morifolium (菊花) --- p.43 / Chapter 2.7.3 --- Taraxacum mongolicum --- p.44 / Chapter 2.7.4 --- Wedelia trilobata (三裂葉蟛蜞菊） --- p.45 / Chapter Chapter 3 --- Materials and Methods / Chapter 3.1 --- Extraction --- p.47 / Chapter 3.1.1 --- Water extraction --- p.47 / Chapter 3.1.2 --- NaOH extraction --- p.48 / Chapter 3.1.3 --- Ethanol precipitation --- p.50 / Chapter 3.1.4 --- Bioactivity guided fractionation --- p.50 / Chapter 3.1.4.1 --- Macroporous resin column (D101) --- p.50 / Chapter 3.1.4.2 --- Silica gel 60 column --- p.51 / Chapter 3.1.5 --- High Performance Liquid Chromatography (HPLC) analysis --- p.53 / Chapter 3.2 --- Characterization --- p.54 / Chapter 3.2.1 --- Chemical tests --- p.54 / Chapter 3.2.1.1 --- Alkaloids --- p.54 / Chapter 3.2.1.1.1 --- Mayer reagent --- p.54 / Chapter 3.2.1.1.2 --- Dragendorff reagent --- p.54 / Chapter 3.2.1.1.3 --- Wagner reagent --- p.55 / Chapter 3.2.1.2 --- Lactones and coumarins --- p.55 / Chapter 3.2.1.2.1 --- Ferric hydroxamine acid test --- p.55 / Chapter 3.2.1.2.2 --- Emersen reagent --- p.56 / Chapter 3.2.1.3 --- Flavonoids --- p.56 / Chapter 3.2.1.3.1 --- Shinoda test --- p.56 / Chapter 3.2.1.3.2 --- Aluminum chloride reagent --- p.56 / Chapter 3.2.1.4 --- Sterols --- p.57 / Chapter 2.2.1.4.1 --- Liebermann-Buchard test --- p.57 / Chapter 3.2.1.4.2 --- Salkowski reaction --- p.57 / Chapter 3.2.1.5 --- Saponins --- p.57 / Chapter 3.2.1.6 --- Carbohydrates --- p.58 / Chapter 3.2.1.6.1 --- Molisch reagent --- p.58 / Chapter 3.2.1.6.2 --- Aniline acetate reaction --- p.58 / Chapter 3.2.1.7 --- Terpenoids --- p.58 / Chapter 3.2.1.7.1 --- Vanillin reagent --- p.58 / Chapter 3.2.1.7.2 --- Carr-price reagent --- p.59 / Chapter 3.2.1.8 --- Anthraquinone --- p.59 / Chapter 3.2.1.8.1 --- Borntrager reaction --- p.59 / Chapter 3.2.1.8.2 --- Magnesium acetate reagent --- p.59 / Chapter 3.2.2 --- X-ray crystallography --- p.60 / Chapter 3.3 --- In vitro Assay --- p.61 / Chapter 3.3.1 --- Cell lines --- p.61 / Chapter 3.3.2 --- Maintenance of cell lines --- p.61 / Chapter 3.3.3 --- In vitro antitumor assay --- p.62 / Chapter 3.3.3.1 --- Trypan blue exclusion method --- p.63 / Chapter 3.3.3.2 --- MTT assay method --- p.65 / Chapter 3.3.3.3 --- Determination of IC50 --- p.66 / Chapter 3.3.4 --- Cytotoxicity assay on normal cell lines --- p.67 / Chapter 3.4 --- In vivo Assay --- p.68 / Chapter 3.4.1 --- Animals --- p.68 / Chapter 3.4.2 --- Maintenance of Sarcoma 180 cell line --- p.68 / Chapter 3.4.3 --- Tumor inoculation --- p.69 / Chapter 3.4.4 --- Preparation of samples --- p.69 / Chapter 3.4.5 --- In vivo antitumor assay --- p.70 / Chapter 3.4.5.1 --- Antitumor effect of WT4-4 on Sarcoma 180 solid tumor --- p.70 / Chapter 3.4.5.2 --- Effect of 5% DMSO in sterile PBS --- p.70 / Chapter 3.4.6 --- Body weight change --- p.71 / Chapter 3.5 --- DNA Agarose Gel Electrophoresis --- p.73 / Chapter 3.6 --- Statistical Analysis --- p.74 / Chapter Chapter 4 --- Results / Chapter 4.1 --- Extraction and characterization of Wedelia trilobata (WT) --- p.76 / Chapter 4.1.1 --- Percentage of yield in extraction and fractionation of WT --- p.76 / Chapter 4.1.2 --- HPLC chromatograms of WT fractions and the purified component (crystal) --- p.77 / Chapter 4.1.3 --- Phytochemical groups of WT4-4 --- p.78 / Chapter 4.1.4 --- Results on X-ray crystallography of the isolated crystal from WT4-4 A --- p.78 / Chapter 4.2 --- In vitro Antitumor Assay --- p.95 / Chapter 4.2.1 --- "Effects ofDIl, DM1, TM1 and WT1 on suspension cancer cell line" --- p.95 / Chapter 4.2.2 --- Effects of WT fractions and the purified component (crystal) on suspension cell lines --- p.95 / Chapter 4.2.3 --- Effects of WT fractions on adhesive cancer cell lines --- p.98 / Chapter 4.2.4 --- Effects of WT4-4 on normal cell lines --- p.98 / Chapter 4.3 --- In vivo Antitumor Effect of WT4-4 --- p.126 / Chapter 4.4 --- Results of DNA Agarose Gel Electrophoresis --- p.130 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- Characterization of Antitumor Fraction and Compound of Wedelia trilobata (WT) --- p.132 / Chapter 5.2 --- Antitumor Effects of WT Fractions and Purified Component (Crystal) --- p.137 / Chapter 5.2.1 --- In vitro assay --- p.137 / Chapter 5.2.2 --- DNA agarose gel electrophoresis --- p.143 / Chapter 5.2.3 --- In vivo assay --- p.144 / Chapter 5.3 --- Further Study --- p.146 / Chapter Chapter 6 --- Conclusion --- p.148 / References --- p.151

Bioactivity-guided isolation of SFE oil led to the identification of another sesquiterpene lactone, 6-O-angeloylprenolin, containing the bioactive alpha, beta-unsaturated cyclopentenone. MTT results showed that CNE cells were more susceptible to 6-O-angeloylenolin than the normal Hs68 cells. Besides, the inhibitory effect of 6-O -angeloylenolin on the CNE cells was slightly stronger than that of cisplatin, the positive control, albeit statistical insignificance. / Both volatile oils prepared by supercritical fluid extraction (SFE) and steam distillation (SD) were evaluated for their anti-NPC potential. Results showed that SFE oil was much stronger than that of SD oil. SFE oil significantly inhibited the growth of CNE cells by dysfunctioning the mitochondria and activating caspases. Gas chromatography-mass spectrometry analysis revealed that the responsible principals in the SFE oil were likely homologues of sesquiterpene lactones. / Centipeda minima (L.) A. Br. (Compositae), a Chinese medicinal herb, is used to treat nasopharyngeal carcinoma (NPC) in the Chinese folk. However, there is a paucity of information on its anticancer activities. In particular, both of its anti-NPC potential and the potent constituents remain elusive. / In this study, the n-hexane fraction of C. minima showed broad spectrum of inhibitory effects on five human cancer cell lines, including the breast carcinoma MCF7 cells, the prostate carcinoma PC-3 cells, the hepatocellular carcinoma Hep G2 cells, the nasopharyngeal cancer CNE cells and the acute promyelocytic leukemia HL-60 cells, with IC 50 values ranging from 6.1 to 47.3 mug/mL. Bioactivity-guided separation of the n-hexane fraction using the CNE cells as the cellular system led to the isolation of a sesquiterpene lactone, 2beta-(isobutyryloxy)florilenalin (IF), which contained the bioactive alpha-methylene-gamma-lactone ring. IF significantly induced CNE cell death with an IC50 value of 3.1 mug/mL. Despite this potency, its effect on the normal Hs68 cells was much weaker, with an IC50 value larger than 50 mug/mL. Its inhibitory effect on the CNE cells ascribed to apoptotic induction as evidenced by the cumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation, caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. Mechanistic study showed that both extrinsic and intrinsic apoptotic pathways were activated. In the extrinsic pathway, IF activated caspase-8, which further induced the activation of caspase-3 and caspase-7. In the intrinsic pathway, IF regulated the expressions of Bcl-2 family proteins, followed by depletion of mitochondrial membrane potential (Delta&PSgr;m), the release of cytochrome c to cytosol, the activation of caspase-9 and other downstream caspases, and finally the induction of apoptosis. / Mechanistic investigation showed that 6-O-angeloylenolin caused cell cycle arrest at S and G2/M phases and induced apoptosis in CNE cells. For the cell cycle arrest, a sharp decrease was found in the expressions of cyclin D1, cyclin D3, cdc25c, and p-cdc25c, with concomitant decrease in CDK4, cyclin A, cyclin E, p-Rb(Ser780), p21Waf1/Cip1, cdc2 and p-cdc2. For the induction of apoptosis, externalization of phosphatidylserine and depletion of Delta&PSgr;m prior to the detection of sub-G1 peak were found. Other apoptotic features including the presence of apoptotic bodies, the activation of caspase-3 activity and the cleavage of PARP were observed. Activation of caspase-8 and caspase-10 was detected. Besides, 6-O -angeloylenolin induced the release of cytochrome c and AIF to cytosol. The former formed apoptosome with caspase-9, further activated the downstream caspase-3 and caspase-7 and cleaved PARP, while the latter was translocated into the nucleus and caused large-scale DNA fragmentation. Failure of the pan-caspase inhibitor, z-VAD-fmk, to interrupt the apoptotic induction by 6-O-angeloylenolin suggested that caspase-independent pathway was involved. 6-O-Angeloylenolin was able to activate Akt, ERK and JNK pathways. But only with the addition of JNK inhibitor (SP600125), significant suppression of the 6-O-angeloylenolin-induced apoptosis was observed, suggesting the involvement of the JNK pathway in the apoptotic pathway. Taken together, this study provided a better mechanistic insight into the potential application of 6-O-angeloylenolin as a candidate for NPC treatment. / Overall, this study revealed that two sesquiterpene lactones, including IF and 6-O-angeloylenolin were found to be responsible for the potent anti-NPC effect of C. minima. This study reiterates the notion that Chinese medicinal herbs traditionally applied to cancer treatment may be good sources of anticancer drug discovery, and sesquiterpene lactone may be a group of noteworthy lead compounds displaying anti-NPC potential. / Su, Miaoxian. / Adviser: Hau Yin Chung. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 100-113). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.