Made available in DSpace on 2014-06-12T15:02:36Z (GMT). No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Universidade Federal de Pernambuco / Ganoderma lucidum é um basidiomiceto muito popular na medicina tradicional chinesa, sendo chamado de Lingzhi ou cogumelo da imortalidade . Estudos revelaram diversas propriedades farmacológicas benéficas deste fungo, sugerindo sua utilização como auxiliar em diversos tratamentos contra doenças humanas. Por não ser muito comum na natureza, o cultivo é a melhor maneira de suprir um mercado cada vez mais amplo. Dentre os vários métodos de cultivo existentes para a fungicultura, a técnica Jun-Cao é considerada a mais promissora para a produção de cogumelos em escala que atenda à demanda crescente. Sabendo que a bioquímica de um organismo pode variar entre diferentes linhagens, e entendendo a necessidade de descobrir novas variedades de G. lucidum, para ampliar as opções de cultivo deste fungo, este trabalho buscou comparar linhagens comerciais chinesas com linhagens brasileiras inexploradas de G. lucidum, para avaliar o valor bioquimico destas. Os resultados mostram que o cultivo combinado de madeira com gramínea, na técnica Jun-Cao, foi mais promissor no desenvolvimento das linhagens tanto em redução no tempo de cultivo quanto na qualidade e quantidade dos cogumelos. A linhagem brasileira (CC-144) destacou-se tanto na velocidade de crescimento, quanto na produtividade de basidiomas e na concentração bioquímica de proteínas, carboidratos, β- glucanas e fenóis, superando as chinesas e se mostrando uma opção para a fungicultura. A análise RAPD mostrou distinção genética entre as linhagens, descartando a possibilidade de ocorrência de clones

Cinco décadas de descubrimientos de agentes antineoplásicos han generado una interesante batería de agentes quimioterapéuticos; sin embargo, aunque resultan útiles, se requieren agentes más efectivos ya que estos compuestos tienen poco impacto en las tasas de supervivencia o en la calidad de vida del paciente, especialmente en algunos tipos de cáncer, tales como el Carcinoma Celular Escamoso de Cabeza y Cuello (CCECC) y el Glioblastoma Multiforme (GBM). El presente trabajo de tesis describe resultados que demuestran la potencialidad terapéutica de dos productos sintéticos (análogos del calcitriol) y un compuesto natural (extracto triterpénico de las esporas de Ganoderma lucidum) como agentes terapéuticos en estos dos tipos de cáncer. El calcitriol (1α, 25(OH)2-vitamina D3) es esencial para el mantenimiento de la salud debido a su participación en un crisol de procesos fisiológicos como el control del metabolismo fosfo-cálcico, el crecimiento celular y el sistema inmune. Este compuesto, además, posee potenciales propiedades preventivas y terapéuticas contra diversas enfermedades hiperproliferativas como el cáncer, pero debido a la hipercalcemia que produce al seguir la posología terapéutica, se ve limitado su empleo como agente antitumoral. Por dichos motivos, se sintetizan análogos del calcitriol con el fin de encontrar alguno/s que presenten efectos antitumorales pero que carezcan de los efectos calcemiantes. Nuestro laboratorio, en conjunto con un grupo de investigación de Química Orgánica de la Universidad de Vigo, diseñamos y sintetizamos análogos del calcitriol que contienen un grupo oxolano o un grupo amida en la cadena lateral con el fin de estudiar su potencialidad terapéutica en cáncer. Como parte de este trabajo de tesis evaluamos si estos análogos podían ser efectivos como agentes antitumorales en CCECC y GBM. Por otra parte, la metodología más empleada en la búsqueda de nuevos principios activos naturales es la bioprospección, que incrementa en diez veces la probabilidad de éxito. En la actualidad, el Instituto Nacional del Cáncer (NCI) de USA está intensificando la búsqueda de metabolitos provenientes de la naturaleza para el descubrimiento de nuevas drogas debido a la gran variedad de compuestos que se encuentran en los productos naturales. Ganoderma lucidum es un reconocido hongo comestible y medicinal, empleado por más de dos mil años por la medicina tradicional China, India y Japonesa. Dentro de los efectos farmacológicos se documentaron sus propiedades antitumorales y contiene más de 400 compuestos bioactivos, entre ellos los triterpenoides (la actividad antitumoral se le atribuye a ellos), polisacáridos, nucleótidos y esteroles. También, como parte de este trabajo de tesis comenzamos a evaluar la actividad antitumoral, para el CCECC y los GBM, del extracto triterpénico de las esporas de este hongo producidos por el Laboratorio de Hongos Comestibles y Medicinales, CERZOS-CCT-Bahía Blanca-CONICET. / Five decades of research of anticancer agents have generated an interesting battery of chemotherapeutic agents; however, even though it is useful, more effective agents are needed since these have little impact in survival rates or in the patient‟s life quality, particularly in certain kinds of cancer, such as head and neck squamous cell carcinoma (HNSCC) and glioblastoma multiforme (GBM). This thesis describes results that demonstrate the therapeutic feasibility of two synthetic products (analogues of calcitriol), and a natural compound (extract triterpene Ganoderma lucidum spores) as therapeutics agents in these two kind of cancer. Calcitriol (1α, 25(OH)2-vitamin D3) is essential for the maintenance of health due to its participation in a crucible of physiological processes such as the control of phospho-calcic metabolism, cell growth and immune system. Moreover, this compound has potential preventive and therapeutic properties against various hyper proliferative diseases such as cancer, but due to the hypercalcemia that causes the therapeutic dosage, its use as antitumoral agent is limited. For these reasons, analogues of calcitrol were synthesized to evaluate if any of them shows antitumor effects but lacking calcemiantes effects. Our lab together with a research group Organic Chemistry at the University of Vigo designed and synthesized analogues of calcitriol that contain an oxolane or amide group in the side chain with the goal of studying its therapeutic potentiality in cancer. In this thesis we evaluate if these new analogues can be effective as antitumoral agents in CCECC and GBM. Furthermore, the most used methodology in the search of new natural assets is bioprospecting, increasing tenfold the chances of success. At present, the National Cancer Institute (NCI) of USA is putting emphasis on the search of metabolites from nature for drug discovery because of the wide variety of compounds found in nature. Ganoderma lucidum is a recognized edible and medicinal mushroom used for over two thousand years by traditional Chinese, Indian and Japanese medicine. Among the pharmacological effects, its anti-tumor properties were documented and contains over 400 bioactive compounds among them triterpenoids (antitumor activity is ascribed to them), polysaccharides, nucleotides and sterols. In this thesis we also evaluate the antitumoral activity on CCECC and GBM of triterpene extract spores of this fungus produced by the Laboratory of Edible and Medicinal Mushrooms, CERZOS-CCT-CONICET-Bahia Blanca.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2017-10-09T22:55:45Z No. of bitstreams: 1 tha?s_almeida_menezes-disserta??o_vers?o final.pdf: 2377444 bytes, checksum: 041a519afa0a22b3fcde7920a33c77c7 (MD5) / Made available in DSpace on 2017-10-09T22:55:45Z (GMT). No. of bitstreams: 1 tha?s_almeida_menezes-disserta??o_vers?o final.pdf: 2377444 bytes, checksum: 041a519afa0a22b3fcde7920a33c77c7 (MD5) Previous issue date: 2014-09-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Ganoderma lucidum (Curtis) P. Karst is a medicinal fungus (Basidiomycota, Polyporaceae) popularly known as "Lingzhi" in China, "Reishi" in Japan and "Mushroom King" in Brazil, which has been cultivated in several lignocellulosic materials. In the present study, the formation of mycelium as well as the production of ligninolytic enzymes and basidiomata of this species were evaluated in waste fruit shell, leaf and bract endemic palm of the Brazilian semi-arid region, Syagrus coronata (Martius) Beccari (licuri) by solid state fermentation. The optimal cultivation conditions were the same for all three types of substrates tested (pH 6.5, C/N ratio of 40 and 30?C of temperature) and were established from assays in Petri dishes and contour graphic analysis and response surface methodology. The enzymatic activity of laccase and manganese peroxidase were determined using spectrophotometric methods at intervals of 7 days by cultivating the strain in the three substrates selected for 28 days of incubation. The activity of the laccase was determined at 420 nm, using ABTS: 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) as the enzymatic substrate, and the activity of the manganese peroxidase at 610 nm by oxidation of phenol red. The highest peak activity for laccase (13,80 U/L) was found in bract substrate in 14 days of incubation and did not differ significantly from the others at 95% of statistical confidence. That substrate also resulted in the highest peak of activity for manganese peroxidase (14,92 U/L), which only occurred after 28 days of incubation, differing from the previous one at 5% probability. The assay for the production of basidiomata was performed in polypropylene bags. The only substrate that did not promote the formation of the basidiomata was the fruit shell. The biological yield did not differ significantly at 95% confidence in the bract (33,53 g/kg) and leaf (37,48 g/kg) substrates. The biological efficiency also was not statistically different in the bract (3,35%) and leaf (3,75%) substrates. Thus, it is believed that the licuri wastes are potential substrates and can be used in the bioconversion processes for the formation of the mycelium and basidiomata as well for ligninolytic enzymes production in G. lucidum. / Ganoderma lucidum (Curtis) P. Karst ? um fungo medicinal, do Filo Basidiomycota, Fam?lia Polyporaceae, popularmente conhecido como ?Lingzhi? na China, ?Reishi? no Jap?o e ?Cogumelo Rei? no Brasil, que tem sido cultivado em diversos materiais lignocelul?sicos. No presente estudo, foram avaliadas a forma??o de mic?lio, bem como a produ??o de basidiomas e enzimas ligninol?ticas da esp?cie em res?duos de casca de fruto, folha e br?ctea da palmeira end?mica da regi?o semi-?rida brasileira, Syagrus coronata (Martius) Beccari (licuri), por meio de fermenta??o em estado s?lido. As condi??es ?timas de cultivo foram as mesmas para os tr?s tipos de substratos testados (pH 6,5, rela??o C/N de 40 e temperatura de 30?C), sendo estabelecidas a partir do ensaio em placas de Petri e an?lise dos gr?ficos de contorno e superf?cie de resposta. A atividade enzim?tica de lacase e de mangan?s peroxidase foram determinadas empregando m?todos espectrofotom?tricos, em intervalos de 7 dias, atrav?s do cultivo da linhagem nos tr?s substratos selecionados durante 28 dias de incuba??o. A atividade de lacase foi determinada a 420 nm, empregando ABTS: 2,2?-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) como substrato enzim?tico e a de mangan?s peroxidase, a 610 nm, pela oxida??o do vermelho de fenol. O maior pico de atividade para a lacase (13,80 U/L) foi verificado no substrato br?ctea, aos 14 dias de incuba??o, e n?o diferiu significativamente dos demais a 95% de confian?a. Esse substrato tamb?m proporcionou o maior pico de atividade para a mangan?s peroxidase (14,92 U/L), que somente ocorreu aos 28 dias de incuba??o, diferindo estatisticamente dos demais a 5% de probabilidade. O ensaio para a produ??o de basidiomas foi realizado em sacos de polipropileno. O ?nico substrato que n?o promoveu a forma??o de basidiomas foi a casca de fruto de licuri. O rendimento biol?gico n?o diferiu significativamente a 95% de confian?a nos substratos br?ctea (33,53 g/kg) e folha (37,48 g/kg). A efici?ncia biol?gica tamb?m n?o foi estatisticamente diferente nos substratos br?ctea (3,35%) e folha (3,75%). Assim, acredita-se que os res?duos de licuri sejam potenciais substratos e possam ser empregados no processo de bioconvers?o para a forma??o de mic?lio e basidiomas, e tamb?m para a produ??o de enzimas ligninol?ticas de G. lucidum.

Qin, Jing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 162-218). / Abstract also in Chinese. / Acknowledgment --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Table of Content --- p.vi / List of Figures --- p.x / List of Tables --- p.xv / Abbreviations --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ganoderma spp --- p.1 / Chapter 1.1.1 --- Introduction of Ganoderma spp --- p.1 / Chapter 1.1.2 --- Bioactivities of Ganoderma spp --- p.3 / Chapter 1.1.3 --- Endocrine system and breast cancer --- p.11 / Chapter 1.1.3.1 --- Estrogen --- p.11 / Chapter 1.1.3.2 --- Estrogen receptors --- p.12 / Chapter 1.1.3.3 --- Estrogen responsive genes --- p.15 / Chapter 1.1.3.3.1 --- pS2 --- p.15 / Chapter 1.1.3.3.2 --- Progesterone receptor --- p.18 / Chapter 1.1.3.4 --- Androgen --- p.21 / Chapter 1.1.3.5 --- Androgen receptor --- p.23 / Chapter 1.1.3.6 --- Androgen responsive gene --- p.24 / Chapter 1.1.3.6.1 --- Transmembrane prostate androgen-induced RNA --- p.24 / Chapter 1.1.3.6.2 --- Uridine diphosphate glucose dehydrogenase --- p.26 / Chapter 1.1.3.7 --- Breast cancer --- p.26 / Chapter 1.2 --- "Endocrine Disruption of p,p '-DDE" --- p.28 / Chapter 1.2.1 --- Introduction of p´ةp '-DDE --- p.28 / Chapter 1.2.2 --- "p,p '-DDE in environments" --- p.29 / Chapter 1.2.3 --- "p,p '-DDE in human body" --- p.32 / Chapter 1.2.4 --- "p,p '-DDE and reproductive system" --- p.33 / Chapter 1.2.5 --- Endocrine disruptor --- p.35 / Chapter 1.2.6 --- "Action mechanism of p,p '-DDE on endocrine system" --- p.37 / Chapter 1.2.7 --- Apoptosis --- p.39 / Chapter 1.3 --- Food therapy against endocrine disruption --- p.41 / Chapter 1.3.1 --- Food therapy and functional food --- p.41 / Chapter 1.3.2 --- Ganoderma as a Functional food --- p.47 / Chapter 1.3.3 --- Cancer prevention by dietary agents --- p.47 / Chapter 1.3.4 --- Hormone therapy --- p.48 / Chapter 1.3.5 --- Hormone-related properties of Ganoderma spp --- p.50 / Chapter 1.4 --- The aim of the study --- p.51 / Chapter Chapter 2 --- Materials and Methods --- p.52 / Chapter 2.1 --- Ganoderma samples --- p.52 / Chapter 2.2 --- Artificial cultivation of Ganoderma spp --- p.54 / Chapter 2.3 --- Molecular identification of Ganoderma spp --- p.55 / Chapter 2.3.1 --- Extraction of genomic DNA --- p.55 / Chapter 2.3.2 --- Gene-specific polymerase chain reaction (PCR) --- p.56 / Chapter 2.3.3 --- Gel electrophoresis --- p.56 / Chapter 2.3.4 --- Purification of PCR amplified product for sequencing --- p.57 / Chapter 2.3.5 --- Cycle-sequencing --- p.57 / Chapter 2.3.6 --- Sequencing --- p.58 / Chapter 2.3.7 --- Sequence analysis --- p.58 / Chapter 2.4 --- Chemical analyses of Ganoderma spp --- p.59 / Chapter 2.4.1 --- Polysaccharide preparations --- p.59 / Chapter 2.4.2 --- Terpene profile --- p.60 / Chapter 2.4.3 --- Fatty acid profile --- p.60 / Chapter 2.5 --- Anti-oxidation activities --- p.61 / Chapter 2.5.1 --- Superoxide radical scavenging assay --- p.61 / Chapter 2.5.2 --- DPPH radical scavenging assay --- p.62 / Chapter 2.6 --- Anti-proliferation effect on human breast cancer cells --- p.62 / Chapter 2.7 --- Hormone-like effects --- p.63 / Chapter 2.7.1 --- E-screen test --- p.63 / Chapter 2.7.2 --- In vitro estrogen receptors (ERs) competitor binding assays --- p.64 / Chapter 2.7.3 --- "Recombinant yeast cell based ER-, AR- and PGR-responsible promoter assays" --- p.65 / Chapter 2.7.3.1 --- Recombinant yeasts --- p.65 / Chapter 2.7.3.2 --- Growth medium for recombinant yeasts --- p.66 / Chapter 2.7.3.3 --- "ER, AR and PGR assays" --- p.67 / Chapter 2.7.3.4 --- β-Galactosidase assay --- p.67 / Chapter 2.7.4 --- Real time PCR --- p.68 / Chapter 2.8 --- Flow cytometry --- p.71 / Chapter 2.9 --- Comet assay --- p.71 / Chapter 2.10 --- DNA microarray --- p.73 / Chapter 2.10.1 --- Total RNA isolation --- p.73 / Chapter 2.10.2 --- cDNA synthesis --- p.73 / Chapter 2.10.3 --- Preparation of labelled cDNA --- p.74 / Chapter 2.10.4 --- cDNA purification --- p.74 / Chapter 2.10.5 --- Oligo GEArray hybridization --- p.75 / Chapter 2.10.6 --- Chemiluminescent detection --- p.76 / Chapter 2.10.7 --- Data analysis --- p.77 / Chapter Chapter 3 --- Results --- p.78 / Chapter 3.1 --- Analysis of Ganderma spp --- p.78 / Chapter 3.1.1 --- Mycelia and fruiting bodies --- p.78 / Chapter 3.1.2 --- Identification of Ganoderma spp --- p.79 / Chapter 3.1.3 --- Chemical properties of samples --- p.80 / Chapter 3.1.4 --- Anti-oxidation activities --- p.90 / Chapter 3.1.5 --- Anti-proliferation effect on human breast cancer cells --- p.90 / Chapter 3.1.6 --- Hormone-like bioactivities --- p.93 / Chapter 3.1.6.1 --- E-screen test --- p.93 / Chapter 3.1.6.2 --- In vitro estrogen receptors (ERs) competitor binding assays --- p.94 / Chapter 3.1.6.3 --- "Recombinant yeast cell-based ER-, AR- and PGR-responsible promoter assays" --- p.95 / Chapter 3.1.6.4 --- ER- and AR-pathway gene expression by real time PCR --- p.97 / Chapter 3.2 --- "Action mechanism of p,p' -DDE" --- p.99 / Chapter 3.2.1 --- E-screen --- p.99 / Chapter 3.2.2 --- In vitro estrogen receptors (ERs) competitor binding assays --- p.101 / Chapter 3.2.3 --- Recombinant yeast cell based ER- and AR-responsible promoter assays --- p.103 / Chapter 3.2.4 --- ER- and AR-pathway gene expression by real time PCR --- p.106 / Chapter 3.3 --- Ganoderma tsugae mycelia extract against p.p' -DDE --- p.109 / Chapter 3.3.1 --- E-screen test --- p.109 / Chapter 3.3.2 --- ER- and AR-pathway gene expression by real time PCR --- p.110 / Chapter 3.3.3 --- Analysis of cell cycle --- p.112 / Chapter 3.3.4 --- Analysis of DNA damage --- p.114 / Chapter 3.3.5 --- Analysis of sub-G1 peak --- p.117 / Chapter 3.3.6 --- DNA damage and apoptosis relative gene expression by real time PCR --- p.120 / Chapter 3.3.7 --- DNA microarray --- p.121 / Chapter Chapter 4 --- Discussion --- p.131 / Chapter 4.1 --- Analysis of Ganoderma spp --- p.131 / Chapter 4.2 --- Effects of p.p´ة-DDE --- p.144 / Chapter 4.3 --- Protective effects of G. tsugae against p.p' -DDE --- p.151 / Chapter 4.4 --- Further investigation --- p.159 / Chapter 4.5 --- Conclusion --- p.160 / References --- p.162

Made available in DSpace on 2014-12-17T14:13:46Z (GMT). No. of bitstreams: 1 Raquel Terra Agostinho_DISSERT.pdf: 212655 bytes, checksum: afa93fe07c8ba1db5dc83395a1dc586d (MD5) Previous issue date: 2009-09-29 / Clinical evaluations have been made possible with radiobiocomplexes marked with tecnecium-99m (99mTc). Natural or synthetic drugs are able to interfere in the marking of blood structures with 99m Tc. Also, the toxicity of several natural products has been described. The aim of this study was evaluating the effect of an extract of Ganoderma lucidum (Reishi) in the marking of blood constituents with 98m Tc and in the survival of Escherichia coli. Blood samples from Wistar rats were treated with reishi extract. Radiomarking procedure was performed. Samples of plasma (P), blood cells (CS), and insoluble (FI) and soluble (FS) fractions of P and CS were separated and the radioactivity was counted to determine radioactivity percentages (%ATI). Escherichia coli AB1157 cultures were treated with stannous chloride in the presence and absence of the reishi extract. Blood samples and bacterial cultures treated with NaCl 0.9% were used as controls. Data indicated that the reishi extract has significantly altered (p<0,05) the %ATI of P, CS, FI-P, FS-P, FI-CS e FS-CS, as well as it has increased survival of bacterial cultures treated with stannous chloride. Our results suggest that the Reishi extract would be able to present a redox/ chelant action by altering blood constituent marking with 99mTc and by protecting bacterial cultures against stannous chloride-induced oxydating lesions. The study had a multidisciplinary character, with the participation of the following areas of knowledge: Biophysics, Radiobiology, Botanics, Phytotherapy, and Hematology / Avalia??es cl?nicas t?m sido poss?veis com radiobiocomplexos marcados com tecn?cio-99m (99mTc). Drogas naturais ou sint?ticas s?o capazes de interferir na marca??o de estruturas sangu?neas com 99mTc, e tamb?m tem sido descrita a toxicidade de v?rios produtos naturais. O objetivo deste estudo foi avaliar o efeito de um extrato de Ganoderma lucidum (Reishi) na marca??o de constituintes sangu?neos sang??neas com 99mTc e na sobreviv?ncia de Escherichia coli. Amostras de sangue de ratos Wistar foram tratadas com extrato de reishi. O procedimento de radiomarca??o foi realizado. Amostras de plasma (P), c?lulas sang??neas (CS) e fra??es insol?vel (FI) e sol?vel (FS) de P e CS foram separadas e a radioatividade foi contada para determina??o das porcentagens de radioatividade (%ATI).Culturas de Escherichia coli AB1157 foram tratadas com cloreto estanoso na presen?a e aus?ncia do extrato de reishi. Amostras de sangue e culturas bacterianas tratadas com NaCl 0.9% foram usadas como controles. Dados indicaram que o extrato de reishi alterou significativamente (p<0,05) a %ATI de P, CS, FI-P, FS-P, FI-CS e FS-CS, bem como, aumentou a sobreviv?ncia de culturas bacterianas tratadas com cloreto estanoso. Nossos resultados sugerem que o extrato de Reishi poderia apresentar a??o redox/quelante alterando a marca??o de constituintes sang??neos com 99mTc e protegendo culturas bacterianas contra les?es oxidativas induzidas pelo cloreto estanoso. O estudo teve car?ter multidisciplinar com a participa??o das seguintes ?reas do conhecimento: Biof?sica, Radiobiologia, Bot?nica, Fitoterapia e Hematologia

The medical mushroom Ganoderma lucidum has long been used in traditional Chinese medicine and shown effective in the treatment of many diseases including cancer. Here we studied the cytotoxic effects of two natural compounds purified from Ganoderma lucidum, ergosterol peroxide and ganodermanondiol. We found that these two compounds exhibited cytotoxicity not only against fast proliferating cells, but on quiescent, slow-cycling cells. Using a fibroblast cell-quiescence model, we found that the cytotoxicity on quiescent cells was due to induced apoptosis, and was associated with a shallower quiescent state in compound-treated cells, resultant from the increased basal activity of an Rb-E2F bistable switch that controls quiescence exit. Accordingly, we showed that quiescent breast cancer cells (MCF7), compared to its non-transformed counterpart (MCF10A), were preferentially killed by ergosterol peroxide and ganodermanondiol treatment presumably due to their already less stable quiescent state. The cytotoxic effect of natural Ganoderma lucidum compounds against quiescent cells, preferentially on quiescent cancer cells vs. non-cancer cells, may help future antitumor development against the slow-cycling cancer cell subpopulations including cancer stem and progenitor cells.

Aims. The objectives of this study were (i) to isolate and characterize by conventional column chromatography, structurally diverse triterpenes from G. lucidum to serve as chemical markers; (ii) to develop a high performance liquid chromatography (HPLC) method for the quality control and/or standardization of Lingzhi-containing products; (iii) to utilize and optimize operating conditions for the newer extraction technology: supercritical fluid extraction (SFE), in order to maximize yields of bioactive triterpenes, and to reduce time and costs. / Background. The dried fruiting body of Ganoderma lucidum, commonly known as Lingzhi, has been used extensively as a traditional Chinese medicine (TCM) for many centuries not only in China, but also in other countries such as Japan and Korea. In recent years, Lingzhi has also become a popular health supplement in many Western countries. The chemical composition of Lingzhi is complex, but it has been well documented that the lipophilic triterpenoid class of compounds possess a range of biological effects that include antitumor, immunomodulatory, cardiovascular, respiratory and antihepatotoxic activity. A major drawback in TCM research has been the lack of authentic chemical standards, and efficient methods for the extraction and analysis of bioactive fractions and/or single components. Conventional extraction methods for G. lucidum are time-consuming and laborious, and often result in low yields of useful chemical constituents. / Conclusion. This study enabled the development of a method for the simultaneous analysis of structurally diverse triterpenes with remarkably different chromatographic profiles. The isolated triterpenes, as chemical markers, and the HPLC method can readily be used for quality control and/or standardization purposes in evaluating Lingzhi-containing products. Optimization of operating conditions for SFE facilitated the rapid and selective extraction of acidic triterpenes from raw G. lucidum in significantly higher yields. / Methods. Raw material of G. lucidum was extracted with 80% ethanol; subjected to repeated column chromatography to purify triterpenes; and characterized by NMR (1H and 13C) and mass spectroscopy. Isolated lipophilic triterpenes were qualitatively and quantitatively analyzed by reversed-phase HPLC using an ODS column (150 x 4.6 mm) and PDA detection at 256 nm. The assay was validated over appropriate concentration ranges and benzophenone was used as an internal standard. Supercritical fluid extraction of G. lucidum was carried out using a commercial supercritical fluid extractor system Thar, SFE-1000M. Briefly, the raw powder of G. lucidum was soaked in ethanol containing 10% aqueous ammonia for 30 minutes prior to extraction. Extractions were performed at temperatures of 40, 50 and 60&deg;C; and pressures of 200, 250, 300 and 350 bar; 5% ethanol was used as the co-solvent; and the flow rate of CO 2 was set at 20 g/min. / Results. Eight compounds were isolated and identified from G. lucidum: four triterpenes; namely, lucidenic acid N, ganoderic acid B, ganodermanontriol, and ganodermadiol; two steroids; ergosterol-7, 22-dien-3beta-ol and ergosterol peroxide; and two fatty acids, oleic acid and tetracosanoic acid. The four triterpenes were utilized as chemical markers, and the developed HPLC method was able to simultaneously analyze the structurally diverse components with good resolution. The total analysis run time was 110 min, and retention times (tR) were 14.88, 18.96, 63.88 and 90.73 min respectively, and the eluting system was a mixture of three solvents, methanol (A), acetonitrile (B) and 2% acetic acid solution (C): 0-22 min, 5% A, 25% B and 70% C; 22-85 min, gradient elution, the ratio changed gradually to 5% A, 80% B and 15% C; 85-110 min, gradient elution, the ratio changed gradually to 5% A, 85% B and 10% C. The validated HPLC method and the isolated chemical markers were effectively applied to determine the triterpenoid contents in a variety of commercial Lingzhi products. Supercritical fluid extraction conditions of: pressure 300 bar and temperature 50&deg;C, gave the highest yields of triterpene-containing extracts. HPLC analysis of the SFE extracts showed predominantly acidic triterpenes such as lucidenic acid N and ganoderic acid B. / Hong Xin. / "December 2006." / Advisers: Ho Yee Ping; Albert H. L. Chow. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5968. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 149-175). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Luk Wing Yan Vivien. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 207-239). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abstract --- p.iii / 摘要 --- p.vi / Contents --- p.viii / List of Figures --- p.xiv / List of Table --- p.xxv / Abbreviations --- p.xxv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ganoderma spp --- p.1 / Chapter 1.2 --- Bioactive components of Ganoderma spp --- p.3 / Chapter 1.2.1 --- Lingzhi polysaccharide --- p.3 / Chapter 1.2.2 --- Terpenes --- p.4 / Chapter 1.3 --- Ganoderma spp. as Chinese traditional medicine --- p.5 / Chapter 1.4 --- Artificial hybridisation of Ganoderma luciudm and G. tsugae --- p.6 / Chapter 1.4.1 --- Protoplast isolation and fusion of Ganoderma tsugae and G. lucidum --- p.8 / Chapter 1.5 --- Breast Cancer --- p.8 / Chapter 1.5.1 --- Anti-tumor effects of natural substances against breast cancer cell MCF-7 --- p.9 / Chapter 1.5.2 --- Anti-tumor effects of natural substances against breast cancer cell MDA-MB-231 --- p.11 / Chapter 1.5.3 --- Anti-proliferation of cancer --- p.12 / Chapter 1.5.3.1 --- Cell cycle arrest --- p.12 / Chapter 1.5.3.2 --- Cell death --- p.13 / Chapter 1.5.4 --- Anti-proliferation assays --- p.17 / Chapter 1.5.4.1 --- MTT assay --- p.17 / Chapter 1.5.4.2 --- Trypan blue cell viability assay --- p.18 / Chapter 1.5.4.3 --- BrdU assay --- p.18 / Chapter 1.6 --- Endocrine system and hormones --- p.19 / Chapter 1.6.1 --- Estrogen --- p.23 / Chapter 1.6.2 --- Estrogen receptors --- p.24 / Chapter 1.6.3 --- Estrogen action --- p.29 / Chapter 1.6.4 --- Estrogenicity assays --- p.32 / Chapter 1.6.4.1 --- Recombinant yeast assay --- p.33 / Chapter 1.6.4.2 --- E-screen assay --- p.35 / Chapter 1.6.4.3 --- Estrogen receptor competitor binding assay --- p.36 / Chapter 1.6.4.4 --- Endogenous estrogen-regulated gene expression assay --- p.39 / Chapter 1.6.4.4.1 --- Transforming growth factor --- p.39 / Chapter 1.6.4.4.2 --- Monoamine oxidase A --- p.40 / Chapter 1.6.4.4.3 --- pS2 --- p.40 / Chapter 1.6.4.5 --- Uterotrophic assay --- p.41 / Chapter 1.6.4.6 --- Comparison of in vitro and in vivo assay --- p.42 / Chapter 1.7 --- Aim of study --- p.45 / Chapter 1.7.1 --- Objectives --- p.45 / Chapter Chapter 2 --- Materials and Methods --- p.47 / Chapter 2.1 --- Fungal culture --- p.47 / Chapter 2.2 --- Artificial hybridization of Ganoderma tsugae and G. lucidum --- p.47 / Chapter 2.2.1 --- Protoplast isolation of Ganoderma tsugae and G. lucidum --- p.47 / Chapter 2.2.2 --- Protoplast fusion of Ganoderma tsugae and G. lucidum --- p.48 / Chapter 2.3 --- Screening and selection of hybrid ´Ø --- p.49 / Chapter 2.3.1 --- Temperature screening --- p.49 / Chapter 2.3.2 --- DNA fingerprint by Arbitarily-primed polymerase chain reaction --- p.49 / Chapter 2.3.2.1 --- Extraction of genomic DNA --- p.49 / Chapter 2.3.2.2 --- Arbitrarily-primed polymerase chain reaction --- p.50 / Chapter 2.3.2.3 --- Gel electrophoresis --- p.51 / Chapter 2.4 --- Confirmation test --- p.51 / Chapter 2.4.1 --- Somatic incompatibility test --- p.51 / Chapter 2.4.2 --- DNA fingerprinting by specific polymerase chain reaction --- p.52 / Chapter 2.4.2.1 --- Specific Polymerase Chain Reaction (PCR) --- p.52 / Chapter 2.4.2.2 --- Purification of PCR products --- p.52 / Chapter 2.4.2.3 --- Cycle-sequencing --- p.53 / Chapter 2.3.2.4 --- Sequencing --- p.54 / Chapter 2.3.2.5 --- Sequence analysis --- p.54 / Chapter 2.5 --- Characterization of the selected hybrid --- p.56 / Chapter 2.5.1 --- Scanning electron microscopy (SEM) --- p.56 / Chapter 2.5.1.1 --- Preparation of specimens for scanning electron microscopy --- p.56 / Chapter 2.5.1.2 --- "Cytological studies of pileus, stipe and spores of G. lucidum, G. tsugae and hybrid" --- p.57 / Chapter 2.5.2 --- Temperature effect --- p.57 / Chapter 2.5.3 --- Submerged fermentation --- p.57 / Chapter 2.5.4 --- Fruiting test --- p.58 / Chapter 2.6 --- "Bioactive components of G. lucidum, G. tsugae and hybrid" --- p.58 / Chapter 2.6.1 --- Sample preparation --- p.58 / Chapter 2 6.2 --- Lingzhi polysaccharide --- p.59 / Chapter 2.6.3 --- Terpenes --- p.59 / Chapter 2.7 --- Effect of extracts against breast cancer cell lines --- p.60 / Chapter 2.7.1 --- Cell culture --- p.60 / Chapter 2.7.2 --- Lingzhi Extract preparation --- p.61 / Chapter 2.7.3 --- Optimization of cell density --- p.61 / Chapter 2.7.3.1 --- MTT assay --- p.61 / Chapter 2 7.3.2 --- Trypan blue cell viability assay --- p.62 / Chapter 2.7.3.3 --- BrdU assay --- p.62 / Chapter 2.7.3.4 --- Growth curve of MCF-7 --- p.63 / Chapter 2.7.3.5 --- Growth curve of MDA-MB-231 --- p.64 / Chapter 2.7.4 --- Anti-proliferative effect of extracts on MCF-7 cells --- p.69 / Chapter 2.7.4.1 --- MTT assay --- p.69 / Chapter 2 7.4.2 --- Trypan blue cell viability assay --- p.69 / Chapter 2.7.4.3 --- BrdU assay --- p.70 / Chapter 2.7.5 --- Study of cultured medium effect of biomass and pileus extracts on MCF-7 cells --- p.71 / Chapter 2.7.5.1 --- Cultured medium effect ofbiomass and pileus extracts --- p.71 / Chapter 2.7.6 --- mRNA expression assay (RT-PCR) --- p.71 / Chapter 2.7.6.1 --- Effect of extract on gene expression --- p.71 / Chapter 2.7.6.2 --- Time effect of extract on gene expression --- p.72 / Chapter 2.7.6.3 --- Isolation of RNA --- p.72 / Chapter 2.7.6.4 --- Quantification and qualification of DNA and RNA by spectrophotometry --- p.73 / Chapter 2.7.6.5 --- First strand cDNA synthesis --- p.73 / Chapter 2.7.6.6 --- Amplification of cDNA --- p.74 / Chapter 2.7.7 --- Effect of biomass and pileus lingzhi polysacchandes and terpenes on MCF-7 cells --- p.75 / Chapter 2.7.7.1 --- Effect of reconstitution of lingzhi polysacchande and terpenes on MCF-7 cells --- p.75 / Chapter 2.7.8 --- Effect of biomass and pileus extracts on MDA-MB-231 cells --- p.76 / Chapter 2.8 --- Estrogenicigy assay --- p.76 / Chapter 2 8.1 --- E-screen test --- p.76 / Chapter 2.8.2 --- Estrogen receptor competitor binding assay --- p.77 / Chapter 2.8.3 --- pS2 mRNA expression assay --- p.78 / Chapter 2.9 --- DNA microarray analysis --- p.79 / Chapter 2.9.1 --- mRNA purification --- p.79 / Chapter 2.9.2 --- RT and LPR (Linear Polymerase Reaction) labeling --- p.80 / Chapter 2 9.3 --- pre-hybridization --- p.81 / Chapter 2.9.4 --- Hybridization --- p.82 / Chapter 2.9.5 --- Detection --- p.82 / Chapter 2.9.6 --- Image acquisition and analysis --- p.83 / Chapter Chapter 3 --- Result --- p.84 / Chapter 3.1 --- Artificial hybndization of Ganoderma tsugae and G. lucidum --- p.84 / Chapter 3.1.1 --- protoplast isomation and fusion of Ganoderma tsugae and G. lucidum --- p.84 / Chapter 3.2 --- Screening and selection of hybrid --- p.84 / Chapter 3.2.1 --- Temperature screening --- p.84 / Chapter 3.2.2 --- DNA fingerprint by Arbitrarily-primed polymerase chain reaction --- p.86 / Chapter 3.3 --- Confirmation tests --- p.88 / Chapter 3.3.1 --- Somatic incompatibility test --- p.88 / Chapter 3.3.2 --- DNA fingerprinting by specific polymerase chain reaction --- p.90 / Chapter 3.4 --- Characterization of selected hybrid --- p.100 / Chapter 3.4.1 --- Scanning electron micscropy --- p.100 / Chapter 3.4.2 --- Temperature effect --- p.103 / Chapter 3.4.3 --- Submerged fermentation --- p.105 / Chapter 3.4.4 --- Fruiting test --- p.107 / Chapter 3.5 --- "Bioactive components of G. lucidum, G. tsugae and hybrid" --- p.109 / Chapter 3.5.1 --- Lingzhi polysaccharide --- p.109 / Chapter 3.5.2 --- Terpenes --- p.109 / Chapter 3.6 --- Effect of extracts against breast cancer cell lines --- p.119 / Chapter 3.6.1 --- Anti-proliferative effect of extracts on MCF-7 cells --- p.119 / Chapter 3.6.2 --- Study of medium effect of biomass and pileus extracts on MCF-7 cells --- p.139 / Chapter 3.6.3 --- mRNA expression assay (RT-PCR) --- p.143 / Chapter 3.6.4 --- Effect of biomass and pileus lingzhi polysaccharides and terpenes on MCF-7 cells --- p.150 / Chapter 3.6.5 --- Effect of biomass and pileus extracts on MDA-MB231- cells --- p.159 / Chapter 3.7 --- Estrogenicity assay --- p.166 / Chapter 3.7.1 --- E-screen assay on biomass and pileus extracts --- p.166 / Chapter 3.7.2 --- E-screen assay on biomass and pileus terpenes and lingzhi polysaccharide --- p.166 / Chapter 3.7.3 --- Estrogen receptor competitor binding assay --- p.169 / Chapter 3.7.4 --- pS2 mRNA expression assay --- p.175 / Chapter 3.8 --- DNA microarray analysis --- p.177 / Chapter Chapter 4 --- Discussion --- p.184 / Chapter 4.1 --- Artificial hybridization of Ganoderma tsugae and G. lucidum --- p.184 / Chapter 4.1.1 --- Protoplast isolation and fusion of Ganoderma tsugae and G. luciudm --- p.184 / Chapter 4.1.2 --- Screening and selection of hybrid --- p.184 / Chapter 4.1.3 --- Characterization of the selected hybrid --- p.185 / Chapter 4.1.4 --- "Nature of hybrid, mutant and variant" --- p.189 / Chapter 4.2 --- Effect of extracts against breast cancer cell lines --- p.190 / Chapter 4.2.1 --- Anti-proliferative effect of extracts on MCF-7 cells --- p.190 / Chapter 4.2.2 --- Study of effect of cultured medium of biomass and pileus extracts on MCF-7 cells --- p.193 / Chapter 4.2.3 --- Effect of biomass and pileus extracts on MDA-MB231- cells --- p.194 / Chapter 4.2.4 --- mRNA expression assay (RT-PCR) --- p.195 / Chapter 4.3 --- Estrogenicity --- p.198 / Chapter 4.3.1 --- E-screen assay --- p.198 / Chapter 4.3.2 --- Estrogen receptor competitor binding assay --- p.199 / Chapter 4.3.3 --- pS2 mRNA expression assay --- p.200 / Chapter 4.3.4 --- Ganoderma spp. As hormonal therapy --- p.201 / Chapter 4.4 --- DNA microarray analysis --- p.201 / Chapter 4.5 --- Further investigation --- p.204 / Chapter Chapter 5 --- Conclusion --- p.205 / Chapter Chapter 6 --- Reference --- p.207

Ganoderma lucidum and Cordyceps sinensis are medicinal fungi commonly used in Chinese medicine. The allied species of G. lucidum, G. sinense and G. tsugae as well as the allied species of C. sinensis, C. militaris are also commercially available as health supplements. The present study aimed at evaluating and comparing the biological activities of the different parts of fruiting bodies of Ganoderma species (whole fruiting body, pileus, stipe, spores and spore oil) and Cordyceps species (whole fruiting body, stroma and larva/pupa). / The extracts of C. sinensis and C. mililaris could stimulate secretion in human airway epithelial cell Calu-3, implying the potentials of these extracts to modulate the mucociliary clearance. The potencies of the stimulatory effects of the stroma of fruiting bodies showed stronger effects than the larva/pupa. All parts of C. sinensis and the stroma of C. mililaris could modulate the proliferation of human peripheral blood mononuclear cells and the different potencies of immunomodulatory effects were also observed. / The results demonstrated that the hot water extracts of G. lucidum, G. sinense and G. tsugae possessed antiproliferative effects on human breast cancer cell lines MCF-7 and MDA-MB-231. The extracts from the stipes showed stronger inhibitory activities on cancer cell proliferation than those from pilei. Furthermore, the extracts from the whole fruiting body and stipe of G. lucidum possessed strong antitumor effects on the nude mice bearing MCF-7 xenografts and the BALB/c mice bearing sarcoma S-180 allografts as well as strong immunomodulatory effects in terms of stimulating splenic lymphocyte proliferative responses. The oral administrations of spores and spore oil did not show significant inhibition on MCF-7 xenografts growth but they inhibited sarcoma allografts growth effectively. / The strong biological effects of the stipe of Ganoderma and the stroma of Cordyceps were showed for the first-time. This study sheds light on how the fruiting bodies of Ganoderma or Cordyceps should be used to achieve the most out of their pharmacological properties. This study also demonstrated the novel application of Cordyceps in promoting secretion in human airway submucosal glands, which reinforces the rationale of using this fungus for treating respiratory diseases. / Yue Gar Lee Grace. / "August 2006." / Advisers: Leung Ping Chung; Kwok Pui Fung; Bik San Clara Lau. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1584. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 318-340). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

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