Thesis--Auburn University, 2009. / Abstract. Includes bibliographical references (p. 108-119).

[Abstract]: This thesis describes an in-vivo evaluation of ethanolic extracts of Astragalus membranaceus and fractions derived from this extract administered either orally by gavage or by intraperitoneal injection to Balb/c mice. Total antibody titre was used as an indicator of humoral immune response. Cell mediated immune response was determined using Interferon-γ and Interleukin-12 as indicators. Additionally, mice were vaccinated with a killed Salmonella typhimurium vaccine, previously demonstrated to induce humoral response but not cell mediated immunity, to determine whether the acquired immune response was enhanced or suppressed. Serum was analysed for total antibody titre using an enzyme-linked immunosorbent assay. Serum and splenocyte culture supernatants were analysed for levels of interferon-γ and interleukin-12. No statistically significant differences were observed between groups either orally gavaged or intraperitoneally injected with extracts of Astragalus membranaceus, or orally gavaged with fractions derived from this extract when compared with the control groups.

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Although tremendous efforts have been made to search for other novel growth factors in promoting marrow recovery after irradiation or chemotherapy, there have not been any efficient and safe agents discovered so far. Danggui Buxue Tang (當歸補血湯) as a traditional Chinese herbal decoction, is commonly used for replenishing blood loss in menstruating women, or enhancing erythropoiesis and immune responses in various settings. Our previous study confirmed that Danggui Buxue Tang promotes haematopoiesis and thrombopoiesis both in vitro & in vivo. Recent studies also showed that parenteral Astragalus regulates haematopoiesis in myelosuppressed mice and has protection effect on UV irradiated human dermal fibroblasts. However, astragaloside IV, as the major component of Astragalus, the "Monarch" (君葯) in Danggui Buxue Tang, the bioactivity and its possible mechanism on haematopoiesis remains unclear. My studies showed that astragaloside IV had promoting effect on different lineages of haematopoietic CFUs forming including erythrocytes, granulocytes, monocytes and megakaryocytes both in normal and irradiated mice. In the K562 and CHRF apoptotic model, astragaloside IV exerted proliferation effect and induced K562 into megakaryocytic differentiation. Astragaloside IV up-regulated phosphorylation of ERK and it was abolished by PD98059. Meanwhile, astragaloside IV increased phosphorylated ERK migration into nuclei which enhanced cell survival and differentiation. EGFR inhibitor also attenuated the enhancing effect of astragaloside IV on ERK phosphorylation. It suggested that astragaloside IV is likely to function through EGFR with subsequent activation of ERK1/2 pathway. Furthermore, astragaloside IV also increased Bcl-2/Bax ratio by up-regulating Bcl-2 alone. Bone marrow derived mesenchymal stromal cells are the major supporting cells involved in the haematopoietic microenvironment. My studies demonstrated that astragaloside IV also indirectly enhanced haematopoiesis by stimulating cytokine release from MSCs, especially IL-6, IL-8, MCP-1 and GRO1. I also found that matured and activated population of neutrophils was increased after cultured with mesenchymal stromal cells conditional medium stimulated by astragaloside IV. This finding further supported why there was a significant increment of CFU-GM in vitro culture with murine bone marrow collected from mouse model after astragaloside IV treatment, where MSCs serve as the feeder layer in such system in mice. In conclusion, my studies explored the directly and indirectly dynamic and multiple targeted function of astragaloside IV on haematopoiesis. In addition to activating haematopoietic cells, astragaloside IV also stimulated mesenchymal stromal cells to secret cytokines that could modulate haematopoiesis and up-regulated neutrophil production and maturation. It provided a holistic view on how astragaloside IV induced synergistic effect on haematopoietic cells and mesenchymal stromal cells in the marrow microenvironment. / published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

Uncontrolled cell growth may lead to pathological conditions such as cancer. During the progression of cancer, cancer cells stimulate endothelial cells for angiogenesis to support their growth and migration. Previous studies suggest that Astragalus membranaceus, of which the dried root [Astragali Radix] is used as a traditional Chinese medicine, and its bioactive components, astragalus saponins (AST), astragaloside IV (AS IV) and isoflavonoid calycosin, inhibit cancer growth. The present study aimed to examine whether or not these components inhibit the growth and/or metastasis of colon cancer cells and/or angiogenesis of endothelial cells, and to determine the possible mechanisms involved. The growth of HCT 116 colon cancer cells and human umbilical vein endothelial cells (HUVEC) after 72 hours incubation with AST (1 to 25 μg/ml), AS IV (0.5 to 100 μM) or calycosin (10 to 200 μM) were detected with thiazolyl blue tetrazolium bromide assay. Wound healing migration and tube formation assays were used to examine the metastatic and angiogenic potential of HCT 116 cells and HUVEC. Moreover, the expressions of apoptotic [B-cell lymphoma 2 and procaspase-3] and metastasis/angiogenesis-related proteins [matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF)] were measured with Western immunoblotting. To investigate the potential mechanism(s) through which astragalus components affect the proliferation and/or migration of HCT 116 cells and HUVEC, the activities of mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 MAP kinase (p38) and c-Jun amino-terminal kinases] were studied by measuring the expressions of their phosphorylated and total proteins with Western immunoblotting. Calycosin (200 μM) inhibited the growth of HCT 116 cells without affecting that of HUVEC. While it inhibited the migration of both cell types, it stimulated tube formation only in HUVEC. In HCT 116 cells, calycosin downregulated the expressions of procaspase-3, VEGF, MMP-2 and MMP-9 proteins, inhibited ERK1/2 but activated p38. These effects of calycosin were not observed in HUVEC. Neither AST nor AS IV had any significant effects on the parameters studied in HCT 116 cells. AST also showed no effect in HUVEC; AS IV, at 100 μM, appeared to increase the number of tube formation by HUVEC. In conclusion, the present findings suggest that AST has no significant effect on both cancer and endothelial cells while AS IV may promote angiogenesis without any direct action in colon cancer cells. In colon cancer cells, calycosin induces apoptosis, possibly through activation of caspase-3 and p38, and inhibits metastasis, possibly by downregulating MMP-2 and MMP-9, and inhibiting ERK1/2. However, in endothelial cells, the effect of calycosin is not conclusive as it promotes tube formation but inhibits migration. These findings provide the pharmacological basis for the use of Astragali Radix in the treatment of colon cancer, and the scientific evidence for a therapeutic potential of calycosin in the management of this disorder. Further studies are needed to verify the effect of calycosin on endothelial cells. In order to better mimic the clinical situation, the interaction between cancer and endothelial cells [for example, tumor-induced angiogenesis] needs to be taken into consideration. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

背景黃芪是中醫藥中最常用的補益藥之一，現代研究發現其多糖類成分具有抗疲勞、抗氧他和免疫調節等作用，因此研究黃芪多糖的生理活性已成為研究黃芪藥理作用新的主流方向。由於多糖類物質分子量較大，單糖組成及組成方式多樣，所以對多糖的研究除生理活性外還需探討解析其他學特征。目的初步驗證黃芪多糖對免疫系統的生理活性及其自身他學組成，探討展望未來對黃芪多糖研究的新方向。方法本實驗採用水提醇沉法提取分離除黃芪粗多糖，經除蛋白及透析等操作純忙得到黃百多糖。採用高效凝膠色譜分離法及超高效液相色譜法分別求得黃芪多糖相對分子量大小及其單糖組成成分免疫活性探究使用RAW264.7 細胞系巨噬細胞，以脂多糖為陽性對照，採用MTT 法測試細胞毒性，計算加藥后一氧化氮及細胞因子IL-6 和TNF－α 生成量，評價黃芪多糖的免疫調節作用。結果黃芪多糖相對分子量為108.02kDa(±2.73kDa），由阿拉伯糖、葡萄糖、半乳糖、葡萄糖醛酸和半乳糖醛酸組成。MTT 實驗表明黃芪多糖對細胞無明顯毒性﹔ NO 及細胞因子IL-6 和TNF－α 生成量表明其具有免疫調節功能，且作用強度與黃芪多糖濃度在一定範圍內呈正相關。結論黃芪多糖具有免疫調節活性，但其組成成分較多，他學結構複雜，仍需要進行更多研究探討其作用機制及其他學結構與免疫調節機制的關係。【關鍵詞】黃芪多糖﹔化學組成﹔免疫活性

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No / Croton membranaceus is used by herbalists and traditional healers in Ghana for the management of various cancers, especially prostate cancers. A methanolic extract of the roots showed cytotoxic activities against two cancer cell lines, and bioassay-guided fractionation of this extract revealed that the cytotoxic activity resided mostly in the ethyl acetate fraction. Six compounds were isolated from this fraction, including a new furano-clerodane diterpenoid (1), for which the trivial name crotomembranafuran is suggested. This compound exhibited an IC50 value of 4.1 microgram/mL (10.6 microM) against human prostate (PC-3) cells, providing some support for the traditional use of C. membranaceus in the treatment of cancers

Yip Pui Ying. / Thesis submitted in: November 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 147-184). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgment --- p.v / Abbreviations --- p.vii / Table of contents --- p.viii / List of Figures --- p.xii / List of Tables --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- The importance of characterization of Chinese medicinal materials and the development of Chinese medicine in Hong Kong --- p.1 / Chapter 1.2. --- Methods for characterization of Chinese medicinal materials --- p.5 / Chapter 1.3. --- Molecular characterization of Chinese medicinal materials --- p.8 / Chapter 1.3.1. --- DNA sequencing --- p.9 / Chapter 1.3.2. --- DNA fingerprinting --- p.14 / Chapter 1.3.3. --- Nucleic acid hybridization --- p.19 / Chapter 1.4. --- Objectives --- p.20 / Chapter Chapter 2 --- Characterization of Plant and Fungal Materials by rDNA ITS Sequence Analysis --- p.22 / Chapter 2.1. --- Introduction --- p.22 / Chapter 2.2. --- Materials and Methods --- p.22 / Chapter 2.2.1. --- Chinese medicinal materials used in this study --- p.22 / Chapter 2.2.1.1. --- Plants and fungi for interspecific ITS study --- p.22 / Chapter 2.2.1.2. --- Plant for intraspecific ITS study and locality study --- p.33 / Chapter 2.2.2. --- Extraction of total DNA --- p.35 / Chapter 2.2.3. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.35 / Chapter 2.2.4. --- Purification of PCR products --- p.38 / Chapter 2.2.5. --- Cloning using pCR-Script´ёØ Amp SK(+) Cloning Kit --- p.38 / Chapter 2.2.5.1. --- Polishing --- p.38 / Chapter 2.2.5.2. --- Ligation of inserts into pCR-Script´ёØ Amp SK(+) cloning vector --- p.38 / Chapter 2.2.5.3. --- Transformation --- p.40 / Chapter 2.2.5.4. --- PCR screening of white colonies --- p.40 / Chapter 2.2.5.5. --- Purification of PCR screening products --- p.41 / Chapter 2.2.6. --- Sequencing of ITS regions --- p.41 / Chapter 2.2.6.1. --- Cycle sequencing reaction --- p.41 / Chapter 2.2.6.2. --- Purification of sequencing extension products --- p.41 / Chapter 2.2.6.3. --- Electrophoresis by genetic analyzer --- p.42 / Chapter 2.2.7. --- Sequence analysis and alignment --- p.42 / Chapter 2.3. --- Results --- p.42 / Chapter 2.3.1. --- Extraction of total DNA --- p.42 / Chapter 2.3.2. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.44 / Chapter 2.3.2.1. --- Interspecific ITS study --- p.44 / Chapter 2.3.2.2. --- Intraspecific ITS study --- p.46 / Chapter 2.3.3. --- Sequence analysis and alignment --- p.47 / Chapter 2.3.3.1. --- Interspecific ITS study --- p.47 / Chapter 2.3.3.2. --- Intraspecific ITS study --- p.56 / Chapter 2.4. --- Discussions --- p.60 / Chapter 2.4.1. --- rDNA regions used for studying Chinese medicinal materials --- p.60 / Chapter 2.4.2. --- The results agreed with previously published works --- p.60 / Chapter 2.4.3. --- Explanation of interspecific results within the Ganoderma genus --- p.60 / Chapter 2.4.4. --- Implications from interspecific comparisons --- p.60 / Chapter 2.4.5. --- Implications from intraspecific comparisons --- p.61 / Chapter Chapter 3 --- .Characterization of Astragalus membranaceus by DNA Fingerprinting / Chapter 3.1 --- Introduction --- p.62 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.2.1 --- Extraction of total DNA --- p.62 / Chapter 3.2.2 --- Generation and detection of DNA fingerprints by AP-PCR --- p.63 / Chapter 3.2.3 --- Analysis of DNA fingerprints --- p.63 / Chapter 3.3 --- Results --- p.63 / Chapter 3.3.1 --- Generation of DNA fingerprints by AP-PCR --- p.63 / Chapter 3.3.2 --- Fingerprint analysis --- p.69 / Chapter 3.4 --- Discussion --- p.85 / Chapter 3.4.1 --- RP-PCR has been used on Chinese medicinal materials --- p.85 / Chapter 3.4.2 --- AP-PCR used instead of RAPD --- p.85 / Chapter 3.4.3 --- Reproducibility and amount of bands --- p.86 / Chapter 3.4.4 --- Alternatives of electrophoresis process --- p.88 / Chapter 3.4.5 --- Explanation of results --- p.88 / Chapter 3.4.6 --- Distinguishing Neimengu and Shanxi samples --- p.89 / Chapter 3.4.7 --- Further studies --- p.90 / Chapter Chapter 4 --- Characterization of Plant and Fungal Materials by DNA-DNA Hybridization on Microarrays --- p.91 / Chapter 4.1 --- Introduction --- p.91 / Chapter 4.2 --- Materials and Methods --- p.92 / Chapter 4.2.1 --- Samples for microarray study --- p.92 / Chapter 4.2.2 --- Extraction of total DNA --- p.95 / Chapter 4.2.3 --- Amplification and sequencing of ITS 1 region of rRNA gene --- p.95 / Chapter 4.2.4 --- Preparation of labeled probe --- p.95 / Chapter 4.2.5 --- Amplification of ITS1 fragments --- p.97 / Chapter 4.2.6 --- Preparation of slides --- p.103 / Chapter 4.2.7 --- Hybridization and washing --- p.104 / Chapter 4.2.8 --- Scanning and data analysis --- p.105 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- DNA extraction --- p.105 / Chapter 4.3.2 --- Amplification and sequencing of ITS1 region of rRNA gene --- p.107 / Chapter 4.3.3 --- Preparation of labeled probe and amplification of ITS1 fragments… --- p.112 / Chapter 4.3.4 --- Preparation of slides --- p.112 / Chapter 4.3.5 --- Scanning and data analysis --- p.116 / Chapter 4.4 --- Discussion --- p.134 / Chapter 4.4.1 --- Implications --- p.134 / Chapter 4.4.2 --- Applying the findings --- p.134 / Chapter 4.4.3 --- Ways to maximize specificity --- p.137 / Chapter 4.4.4 --- Optimisation --- p.138 / Chapter 4.4.5 --- Microarray may be more advantageous over sequencing --- p.138 / Chapter Chapter Five --- General Discussion and Summary --- p.140 / Chapter 5.1. --- Objectives of this study --- p.140 / Chapter 5.2. --- rDNA ITS sequencing --- p.140 / Chapter 5.2.1. --- Description of the approach and summary of the results --- p.140 / Chapter 5.2.2. --- Implications from the results --- p.140 / Chapter 5.2.3. --- Advantages and limitations of DNA sequencing --- p.141 / Chapter 5.3. --- AP-PCR fingerprinting --- p.141 / Chapter 5.3.1. --- Description of the approach and summary of the results --- p.141 / Chapter 5.3.2. --- Advantages and limitations of DNA fingerprinting --- p.142 / Chapter 5.4. --- DNA-DNA hybridization on microarrays --- p.143 / Chapter 5.4.1. --- Description of the approach and summary of the results --- p.143 / Chapter 5.4.2. --- Implications from the results --- p.143 / Chapter 5.4.3. --- Advantages and limitations of DNA hybridization on microarrays. --- p.144 / Chapter 5.5. --- Overall summary --- p.144 / Chapter 5.6. --- Future studies --- p.146 / References --- p.147 / Appendix --- p.185