激素性骨壞死是由於經常使用脈衝性激素處理非骨科性問題引起的一種常見的骨科疾病。在組織病理學上，激素性骨壞死指骨死亡，血管內血栓閉塞和血管外骨髓脂肪沉積會引起缺血導致骨修復不足。上游的分子細胞病理學機制研究表明間充質幹細胞細胞池活性下降，成骨細胞凋亡和骨小梁基質退變導致的不充分修復是激素性骨壞死發生的重要因素。 / 間充質幹細胞是骨髓的基質組成部分，具有分化成多種細胞的潛能。最近的研究表明，激素性骨壞死可能是骨細胞和/或間質幹細胞病變引起的一種疾病。研究發現，在接受類固醇治療而發生骨壞死的病人中，骨髓間充質幹細胞活性下降和分化潛能發生改變。在骨髓細胞中，激素能夠誘導脂肪發生。盛輝等發現來源於激素性骨壞死兔子中的間充質幹細胞成脂分化增強，謝新薈等進一步發現發生激素性骨壞死的兔子，骨缺損修復延遲，這可能是由激素導致的間充質幹細胞潛能發生改變引起的。綜合以上研究表明，間充質幹細胞在骨壞死發生和修復過程中起著重要作用。我們之前報導過淫羊藿黃酮（EFs）的腸代謝產物淫羊藿素Icaritin通過抑制血栓的形成和脂肪沉澱預防激素性骨壞死。最近，我們把Icaritin整合到聚乳酸聚乙醇酸共聚物/磷酸三鈣（PLGA/TCP）支架材料中，形成PLGA/TCP/Icaritin複合支架材料。我們發現PLGA/TCP/Icaritin複合材料可以促進激素性骨壞死骨缺損的修復，肌肉移植發現PLGA/TCP/Icaritin也能促進新生血管的發生。我們也發現單純PLGA/TCP複合材料也能夠促進激素性骨壞死骨缺損的修復，但是潛在的機制尚不清楚。 / 骨是一個高度血管化的組織，依賴於血管和骨細胞密切的時空連結維持骨骼的完整性。因此，血管生成在骨骼發育和骨折修復過程中發揮著舉足輕重的作用。血管為骨的發育和再生提供氧氣，為基質輸送刺激間充質細胞特異性成骨的重要信號，另一方面，骨為血管生成輸送生長因數和細胞。 / 本論文分為以下四個主要部分： / 第一部分: 研究Icaritin對人源間充質幹細胞分化的作用及其機制。流式細胞分選鑒定結果表明我們使用的人源間充質幹細胞能夠特異表達間充質幹細胞表面標誌物。MTT實驗結果顯示Icaritin不影響間充質幹細胞的增殖；分化實驗表明Icaritin在沒有成骨誘導試劑存在的情況下無法影響間充質幹細胞的分化。在成骨誘導試劑存在的情況下，Icaritin促進間充質幹細胞成骨分化，抑制其成脂分化；即時螢光實時定量聚合酶鏈式擴增（RT-PCR）結果顯示Icaritin在間充質幹細胞分化過程中上調成骨基因的表達，下調成脂基因表達。進一步研發發現在成骨分化過程中，Icaritin能夠促進BMP2和beta-catenin 蛋白的表達，而BMP2抑制劑Noggin能夠能夠逆轉Icaritin促進的成骨發生。這些發現表明Icaritin能夠促進而非誘導間充質幹細胞的成骨分化，Icaritin調解間充質幹細胞成骨分化具有BMP2信號通路依賴性。 / 第二部分: 評估激素性骨壞死兔源間充質幹細胞的分化潛能及Icaritin 對異常分化的間充質幹細胞分化潛能的影響。結果表明Icaritin促進正常兔源間充質幹細胞的成骨分化，抑制其成脂分化。激素性骨壞死兔源間充質幹細胞的成骨分化潛能降低，成脂分化升高；而Icaritin能夠劑量依賴性地部分恢復降低的成骨分化潛能，抑制升高的成脂分化活性。激素性骨壞死兔源間充質幹細胞的增殖活性也下降但是不能被Icaritin恢復。Icaritin對激素性骨壞死兔源間充質幹細胞中下降的VEGF的表達無影響。這些發現顯示間充質幹細胞的分化潛能在激素性骨壞死發生過程中遭到破壞，但是能夠被Icaritin部分恢復。 / 第三部分: 評估Icaritin對體外成血管的影響。我們對Icaritin對人臍帶靜脈內皮細胞（HUVECs）的增殖、遷移、管狀結構形成及成血管相關基因的表達的影響進行了檢測。結果表明Icaritin不影響HUVECs的增殖、遷移和管狀結構的形成；RT-PCR結果顯示Icaritin對HUVECs中的VEGF, HIF1a, FGF2 and TGF-beta表達也沒有影響。這些發現表明Icaritin在體外並不能直接作用于血管生成。結果謝新薈和陳詩慧等人的體內研究結果可以推測在骨缺損修復過程中，Icaritin通過促進成骨間接促進血管生成。 / 第四部分: 主要研究Icaritin及複合生物材料在體外體內對間充質幹細胞歸巢的影響。結果表明Iaritin能夠促進間充質幹細胞的遷移並上調血管細胞黏附分子1（VCAM1）的表達。複合材料PLGA/TCP和PLGA/TCP/Icaritin在體外培養的條件下能夠募集間充質幹細胞到材料周圍及進入材料。間充質幹細胞體外用修飾性超順磁性氧化鐵（SPIO@SiO₂-NH₂）納米顆粒標記後，其分化潛能依然保留，增殖和潛能能力稍微下降。兔激素性骨壞死造模完成後，股骨遠端髓芯減壓壞死骨缺損手術，PLGA/TCP和PLGA/TCP/Icaritin複合材料植入缺損孔道，同時把SPIO@SiO₂-NH₂標記的間充質幹細胞注射到距離缺損區20毫米的骨髓腔內。結果顯示只有標記的間充質幹細胞植入而沒有材料植入時，缺損區被脂肪細胞充滿，並沒有標記的間充質幹細胞出現，而在缺損區附近和遠離缺損區的部位有標記的間充質幹細胞出現。同時植入PLGA/TCP複合材料和標記的間充質幹細胞時，標記的間充質幹細胞出現在缺損區的材料中，在缺損區附近沒有標記的間充質幹細胞出現，而在遠離缺損區的部位，有標記的間充質幹細胞出現。同時植入PLGA/TCP/Icaritin和標記的間充質幹細胞時，得到跟植入PLGA/TCP複合材料和標記的間充質幹細胞相似的結果，但是在缺損區域，SPIO陽性的間充質幹細胞數目在PLGA/TCP和PLGA/TCP/Icaritin組別中並未發現有顯著性差異。以上發現表明Icaritin和PLGA/TCP複合材料能夠在體外和體內促進間充質幹細胞的歸巢。 / 綜上所述，複合支架材料PLGA/TCP/Icaritin通過調節間充質幹細胞的歸巢和分化促進激素性骨壞死骨缺損的修復。Icaritin通過BMP2和Wnt/beta-catenin通路調解間充質幹細胞的成骨分化。這是首次研究發現Icaritin及PLGA/TCP支架材料影響骨缺損修復過程中幹細胞歸巢，但是分子細胞生物學機制還需要進一步的研究。 / Steroid-associated osteonecrosis (SAON) is a common orthopaedic problem as the pulsed steroids are frequently prescribed for the treatment of non-orthopaedic medical conditions. Histopathologically, SAON refers to death of bone. Intravascular thrombus occlusion and extravascular marrow lipid deposition cause ischemia, which leads to an inadequate repair of the bone. Recent study revealed upstream pathological mechanism at cellular and molecular level. The decrease in activity of mesenchymal stem cell (MSC) pool, apoptosis of osteocytes, and trabecular bone matrix degeneration may cause bone inadequate repair, a key pathological feature found in SAON. / MSCs are the stromal component of bone marrow (BM) and have the potential to differentiate into several cell types. Recent studies have suggested that SAON may be a disease of bone cells and/or MSCs. With corticosteroid therapy in patients, the MSCs activity decreased and differentiation potential changed. Steroids have been also shown to produce adipogenesis in bone-marrow cells. It has been found adipogenesis of MSCs from SAON rabbits elevated (Sheng et al., 2007a) and bone defect repair was delayed in rabbits with SAON (Xie et al., 2011), this may be caused by altered MSCs potentials. All these findings imply MSCs play a vital role in SAON development and bone defect repair. It had been reported that Icaritin, an intestinal metabolite of Epimedium-derived avonoids (EF) reduced SAON incidence with inhibition of both thrombosis and lipid deposition (Zhang et al., 2009a). More recently, we found integrating Icaritin into PLGA/TCP to form PLGA/TCP/Icaritin composite scaffold could promote SAON bone defect repair and more neovascularization formed in an intramuscular implantation model, and further found PLGA/TCP scaffold only also could promote SAON bone defect repair in rabbits (Wang et al., 2012a). But the underlying mechanism remains unclear. / Bone is a highly vascularized tissue reliant on the close spatial and temporal connection between blood vessels and bone cells to maintain skeletal integrity. Angiogenesis thus plays a pivotal role in skeletal development and bone fracture repair. The vasculature supplies oxygen to developing and regenerating bone and also delivers critical signals to the stroma that stimulate MSC specification to promote bone formation and repair. On the other hand, bone also supplies growth factors and cells for angiogenesis. The content of this thesis is divided into the following four major parts: / Part I: to study the effect and molecular mechanism of Icaritin on the differentiation of human bone marrow-derived MSCs. Human MSC was identified first by flow cytometery and result showed our cultured human MSC expressed standard surface markers of MSCs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation ability of MSCs was not affected by Icaritin. Differentiation assay showed that without oseteogenic supplements (OS), Icaritin had no effect on osteogenic differentiation of MSCs. With presence of OS, Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs. Real- time polymerase chain reaction (RT-PCR) showed that Icaritin up-regulated osteoblastic marker genes expression during osteogenic differentiation of MSCs and inhibited adipogenic gene expression. Further studies showed that Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis. All these findings indicated Icaritin possessed osteopromotive but not osteoinductive potentials during the differentiation of MSCs. Icaritin regulated osteogenic differentiation of MSCs in BMP2 pathway dependent manner. / Part II: to evaluate the differentiation potential of MSCs derived from rabbit with SAON and the effect of Icaritin on the altered differentiation of MSCs. The results showed that Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs derived from normal rabbit. Osteogenic differentiation potential of mesenchymal stem cells derived from rabbit with SAON declined and Icaritin partly rescued the declined osteogenic differentiation potential in dose-dependent manner. Adipogenic differentiation potential of MSCs derived from rabbit with SAON enhanced while the enhanced adipogenesis could be depressed by Icaritin. The proliferation ability of MSCs derived from rabbit with SAON declined while could not be rescued by Icaritin. VEGF expression decreased in MSCs derived from rabbit with SAON but its expression could not be influenced by Icaritin. These findings showed that the differentiation potential of MSCs destroyed during SAON development and this potential could be partially restored by Icaritin. / Part III: to evaluate the in vitro angiogenic effect of Icaritin. The proliferation, migration and tube formation ability of human umbilical vein cells (HUVECs) were detected. The results showed that Icaritin did not affect HUVECs proliferation, migration and tube-like structure formation of HUVECs. Real time PCR showed that VEGF, HIF1a, FGF2 and TGF-beta expression in HUVECs was not changed when HUVECs were treated by Icaritin. These data indicated Icaritin did not directly impact angiogenesis in vitro. Combined with in vivo findings, we supposed Icaritin promoted angiogenesis through its enhanced osteogenesis during bone defect repair. / Part IV: to study Icaritin and scaffold impact on stem cell homing in vitro and in vivo. It was found Icaritin promoted the migration of rabbit MSCs and increased vascular cell adhesion molecule 1 (VCAM1) expression. Composite scaffolds PLGA/TCP and PLGA/TCP/Icaritin could recruit rabbit MSCs under in vitro culture condition. When labeled with SPIO@SiO₂-NH₂, the differentiation potential of rabbit MSCs retained while proliferation and migration ability of rabbit MSCs declined. Two weeks after SAON establishment, PLGA/TCP and PLGA/TCP/Icaritin scaffolds were implanted into the bone tunnel after core-decompression in initial necrotic bone defect in rabbits with SAON, immediately with SPIO@SiO₂-NH₂ labeled MSCs injected into bone marrow cavity locally. The results showed that without scaffold implantation, the tunnel was filled with fat cells and fibrotic tissues and there was no label MSC in the tunnel while there were more labeled cells appeared in bone marrow near the tunnel than far away the tunnel, with both PLGA/TCP and PLGA/TCP/Icaritin implantation, the labeled MSCs migrated into scaffold after its implantation into the bone tunnel while there was no labeled cell next to the tunnel but some were shown away from the tunnel. No significant difference was found in SPIO positive MSCs in bone tunnel between PLGA/TCP and PLGA/TCP/Icaritin group. The findings indicated that at least PLGA/TCP scaffold itself promoted MSCs homing in vitro and in vivo where the released icaritin could execute its osteopromotive effects. / In summary, the composite scaffold PLGA/TCP/Icaritin enhanced bone defect repair in rabbit with SAON by promoting homing and osteogenesis of MSCs. Icaritin promoted osteogenic differentiation of MSCs through BMP2 mediated signal pathway, such as Wnt/beta-catenin signal pathway. It is first time to report that PLGA/TCP scaffold promoted MSCs homing during bone defect repair, but underlying molecular and cellular mechanism need to be further studied. / 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. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yao, Dong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 137-158). / Abstract also in Chinese; some appendixes also in Chinese. / ACKNOWLEDGEMENTS --- p.i / TABLE OF CONTENTS --- p.iii / ABSTRACT (IN ENGLISH) --- p.x / ABSTRACT (IN CHINESE) --- p.xiv / FLOWCHART --- p.xviii / LIST OF PUBLICATIONS --- p.xix / LIST OF ABBREVIATIONS --- p.xxi / LIST OF FIGURES --- p.xxiv / Chapter CHAPTER 1: --- Introduction --- p.1 / Chapter 1 --- Osteonecrosis --- p.2 / Chapter 1.1. --- Etiology --- p.2 / Chapter 1.2. --- Anatomy of femoral head --- p.3 / Chapter 1.3. --- Pathogenesis --- p.4 / Chapter 1.3.1. --- Intraosseous hypertension (Compartment Syndrome of Bone --- p.4 / Chapter 1.3.2. --- Intraosseous hypertension (Compartment Syndrome of Bone) --- p.4 / Chapter 1.3.3. --- Coagulation --- p.5 / Chapter 1.4. --- Development stages of osteonecrosis --- p.5 / Chapter 2. --- Steroids-associated osteonecrosis --- p.11 / Chapter 2.1. --- Epidemiology --- p.12 / Chapter 2.2. --- Histopathology --- p.12 / Chapter 2.3. --- Etiopathogenesis --- p.13 / Chapter 2.3.1. --- Steroid and fat metabolism --- p.14 / Chapter 2.3.2. --- Steroid and endothelial cells --- p.15 / Chapter 2.3.3. --- Steroid and coagulation --- p.16 / Chapter 2.3.4. --- Steroid and angiogenesis --- p.17 / Chapter 2.4. --- Steroid and mesenchymal stem cells (MSCs) --- p.18 / Chapter 2.5. --- Treatment strategies for SAON --- p.18 / Chapter 2.5.1. --- Prevention --- p.19 / Chapter 2.5.2. --- Nonoperative treatment --- p.19 / Chapter 2.5.3. --- Operative treatment --- p.19 / Chapter 2.5.3.1. --- Core decompression strategy --- p.20 / Chapter 2.5.3.2. --- Tissue engineering approach --- p.22 / Chapter 3. --- Epimedium-derived flavonoids (EFs) --- p.22 / Chapter 3.1. --- Icaritin -Intestinal metabolism of EFs --- p.24 / Chapter 3.1.1. --- Anti-tumor activity --- p..25 / Chapter 3.1.2. --- Neuroprotective effects --- p.25 / Chapter 3.1.3. --- Embryonic stem cells differentiation --- p.25 / Chapter 3.1.4. --- Osteogenic differentiation --- p.26 / Chapter 4. --- Poly lactic-co-glycolic acid / tricalcium phosphate (PLGA/TCP) scaffold --- p.26 / Chapter 5. --- PLGA/TCP/Icaritin --- p.28 / Chapter 6. --- Hypothesis of this study --- p.28 / Chapter 7. --- Objective --- p.29 / Chapter CHAPTER 2: --- The effect of phytomolecule Icaritin on differentiation of human mesenchymal stem cells in vitro --- p.30 / Chapter 1. --- Introduction --- p.31 / Chapter 2. --- Material and Methods --- p.33 / Chapter 2.1. --- Ethics --- p.33 / Chapter 2.2. --- Reagents and cell culture --- p.33 / Chapter 2.3. --- Surface phenotypes of human BM-MSCs --- p.33 / Chapter 2.4. --- Osteogenic and adipogenic differentiation of human BM-MSCs treated with Icaritin --- p.34 / Chapter 2.5. --- MTT assay for proliferation of BM-MSCs --- p.34 / Chapter 2.6. --- ALP staining --- p.35 / Chapter 2.7. --- ALP activity assay --- p.35 / Chapter 2.8. --- Alizarin Red S staining --- p.35 / Chapter 2.9. --- Oil Red O staining --- p.35 / Chapter 2.10. --- Ribonucleic acid (RNA) isolation --- p.36 / Chapter 2.11. --- Reverse transcription --- p.36 / Chapter 2.12. --- Real time polymerase chain reaction (RT-PCR) --- p.37 / Chapter 2.13. --- Western blotting --- p.37 / Chapter 2.14. --- Osteogenetic analysis of human MSCs after the addition of BMP2 inhibitor Noggin --- p.39 / Chapter 2.15. --- Statistical analysis --- p.39 / Chapter 3. --- Results --- p.40 / Chapter 3.1. --- Characterization of surface phenotypes of human BM-MSCs --- p.40 / Chapter 3.2. --- Icaritin had no effect on human mesenchymal stem cells (MSCs) proliferation --- p..41 / Chapter 3.3. --- Icaritin promoted osteogenic differentiation of MSCs in presence of osteogenic supplement --- p.42 / Chapter 3.4. --- Icaritin enhanced mineralization in osteogenic differentiation of MSCs only in presence of osteogenic supplement --- p.44 / Chapter 3.5. --- Icaritin upregulated mRNA expression of osteoblastic marker genes during osteogenic differentiation of MSCs --- p.45 / Chapter 3.6. --- Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis --- p..48 / Chapter 3.7. --- Icaritin inhibited fat droplets formation during adipogenic differentiation of MSCs --- p.50 / Chapter 4. --- Discussion --- p.52 / Chapter 5. --- Conclusion --- p.56 / Chapter CHAPTER 3: --- Icaritin rescued abnormal differentiation potential of MSCs derived from rabbit with SAON --- p.57 / Chapter 1. --- Introduction --- p.58 / Chapter 2. --- Methods and materials --- p.59 / Chapter 2.1. --- SAON model establishment --- p.59 / Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.60 / Chapter 2.3. --- Osteogenic and adipogenic differentiation of rabbit BM-MSCs treated with Icaritin --- p.61 / Chapter 2.4. --- MTT Assay for Proliferation of BM-MSCs --- p.62 / Chapter 2.5. --- ALP Staining --- p.62 / Chapter 2.6. --- ALP Activity Assay --- p.62 / Chapter 2.7. --- Alizarin Red S Staining --- p.62 / Chapter 2.8. --- Oil Red O Staining --- p.63 / Chapter 2.9. --- RNA Isolation --- p.63 / Chapter 2.10. --- Reverse transcription --- p.64 / Chapter 2.11. --- Real time Polymerase chain reaction (RT-PCR) --- p.64 / Chapter 2.12. --- Western blotting performance --- p.65 / Chapter 2.13. --- Statistical analysis --- p.65 / Chapter 3. --- Results --- p.66 / Chapter 3.1. --- The osteogenic differentiation potential declined while adipogenic differentiation ability elevated of MSCs derived from SAON rabbits --- p.66 / Chapter 3.2. --- The dose-dependent effect of Icaritin on osteogenic differentiation enhancement of MSCs from normal and SAON rabbits --- p.68 / Chapter 3.3. --- Icaritin inhibited adipogenic differentiation of MSCs both derived from normal and SAON rabbits --- p..71 / Chapter 3.4. --- PPAR-γ and aP2 proteins expression increased in SAON rabbit while inhibited by Icaritin both in normal and SAON rabbit --- p.74 / Chapter 3.5. --- Proliferation ability of MSCs derived from SAON rabbit declined and Icaritin had no effect on proliferation both derived from normal and SAON rabbit --- p.75 / Chapter 3.6. --- Icaritin had no effect on the expression of VEGF which decreased in MSCs derived SAON --- p.76 / Chapter 4. --- Discussion --- p.76 / Chapter 5. --- Conclusion --- p.81 / Chapter CHAPTER 4: --- The effect of Icaritin on angiogenesis in vitro --- p.82 / Chapter 1. --- Introduction --- p.83 / Chapter 2. --- Material and Methods --- p.85 / Chapter 2.1. --- Cell culture --- p.85 / Chapter 2.2. --- Proliferation assay --- p.85 / Chapter 2.3. --- Scratch-wound healing assay --- p..86 / Chapter 2.4. --- Migration Assay --- p.86 / Chapter 2.5. --- In vitro Angiogenesis Assay --- p.87 / Chapter 2.6. --- RNA Isolation and Real-time PCR Performance --- p.87 / Chapter 2.7. --- Statistical Analysis --- p.88 / Chapter 3. --- Results --- p.88 / Chapter 3.1. --- Icaritin did not affect HUVECs migration --- p.88 / Chapter 3.2. --- Icaritin had no effect on tube formation on growth factors reduced Matrigel --- p.92 / Chapter 3.3. --- Icaritin had no effect on HUVECs proliferation --- p.94 / Chapter 3.4. --- Icaritin did not change the angiogenesis related gene expression --- p.95 / Chapter 4. --- Discussion --- p.96 / Chapter 5. --- Conclusion --- p.100 / Chapter CHAPTER 5: --- Effect of PLGA/TCP and PLGA/TCP/Icaritin composite scaffolds on stem cell homing during bone defect repair with SAON --- p.101 / Chapter 1. --- Introduction --- p.102 / Chapter 2. --- Material and Methods --- p.106 / Chapter 2.1. --- Preparation of porous PLGA/TCP/Icaritin composite scaffolds --- p.106 / Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.106 / Chapter 2.3. --- Wound healing assay --- p.107 / Chapter 2.4. --- In vitro MSCs recruitment assay of scaffolds --- p.107 / Chapter 2.5. --- MSCs labeling with SPIO@SiO2-NH2 nanoparticle --- p.108 / Chapter 2.6. --- Prussian blue staining --- p.108 / Chapter 2.7. --- MTT assay for SPIO@SiO2-NH2 labeled MSCs --- p.108 / Chapter 2.8. --- Osteogenic and adipogenic differentiation of SPIO@SiO2-NH2 labeled MSCs --- p.109 / Chapter 2.9. --- Real time PCR --- p.109 / Chapter 2.10. --- Animal model establishment --- p.109 / Chapter 2.11. --- Descriptive histology and histomorphometry --- p.110 / Chapter 2.12. --- In vivo magnetic resonance imaging (MRI) of nanoparticle-labeled MSCs --- p.112 / Chapter 2.13. --- Statistical analysis --- p.112 / Chapter 3. --- Results --- p.112 / Chapter 3.1. --- Icaritin promoted MSCs migration in vitro --- p.112 / Chapter 3.2. --- PLGA/TCP and PLGA/TCP/Icaritin recruited MSCs when incubated in vitro --- p.114 / Chapter 3.3. --- Stem cell potentials of MSC after SPIO@SiO2-NH2 labeling --- p.118 / Chapter 3.4. --- PLGA/TCP and PLGA/TCP/Icaritin promoted MSCs homing in vivo --- p.122 / Chapter 4. --- Discussion --- p.126 / Chapter 5. --- Conclusion --- p.136 / Chapter CHAPTER 6: --- Summary of the study and future research --- p.137 / Chapter 1. --- Summary of the study --- p.138 / Chapter 2. --- Limitations and further studies --- p.139 / APPENDIXES --- p.142 / REFERENCES --- p.147

by Kong Lai Ping, Ada. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 171-189). / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.v / TABLE OF CONTENTS --- p.ix / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.1 --- Development of Hematopoietic Stem Cells and Sites of Hematopoiesis --- p.1 / Chapter 1.1.2 --- Role of Cytokines in the Control of Hematopoiesis --- p.3 / Chapter 1.2 --- Leukemia and Cell Differentiation --- p.5 / Chapter 1.2.1 --- Leukemia as Abnormalities in Hematopoietic Cell Development --- p.5 / Chapter 1.2.2 --- Classification and Etiology of Leukemia --- p.6 / Chapter 1.2.3 --- Current Modalities for the Treatment of Leukemia --- p.9 / Chapter 1.2.4 --- Leukemia Cell Lines as In Vitro Models for the Study of Myeloid Leukemia --- p.10 / Chapter 1.2.5 --- Cytokines as Inducers of Myeloid Leukemia Cell Differentiation --- p.12 / Chapter 1.2.6 --- The Murine Myeloid Leukemia Cell Line (WEHI- 3B JCS) as an Experimental Cell Model --- p.13 / Chapter 1.3 --- Flavonoids: Properties and Biological Activities --- p.15 / Chapter 1.3.1 --- Chemical Structure and Classification of Flavonoids --- p.15 / Chapter 1.3.2 --- Occurrence and Distribution of Flavonoids --- p.16 / Chapter 1.3.3 --- Biological Properties and Action Mechanisms of Flavonoids --- p.17 / Chapter 1.3.4 --- Effects of Flavonoids on Leukemia --- p.20 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.23 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.26 / Chapter 2.1.1 --- Cell Lines --- p.26 / Chapter 2.1.2. --- Mice --- p.28 / Chapter 2.1.3 --- Flavonoids --- p.28 / Chapter 2.1.4 --- Recombinant Cytokines --- p.30 / Chapter 2.1.5. --- Physiological Differentiation Inducers ´ؤ Vitamin Analogs --- p.31 / Chapter 2.1.6 --- Monoclonal Antibodies --- p.31 / Chapter 2.1.7 --- "Buffers, Culture Medium and Other Reagents" --- p.33 / Chapter 2.1.8 --- Oligonucleotide Primers and Internal Probes --- p.36 / Chapter 2.1.9 --- Reagents for Cytokine Gene Expression Study --- p.38 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Culture of Tumor Cell Lines --- p.44 / Chapter 2.2.2 --- Determination of Cell Growth and Proliferation --- p.45 / Chapter 2.2.3 --- Colony Assay --- p.46 / Chapter 2.2.4 --- In vivo Tumorigenicity Assay --- p.46 / Chapter 2.2.5 --- Induction of Leukemic Cell Differentiation --- p.47 / Chapter 2.2.6 --- Cell Morphological Study --- p.47 / Chapter 2.2.7 --- Assessment of Differentiation Associated Characteristics --- p.48 / Chapter 2.2.7.1 --- Nitroblue Tetrazolium (NBT) Reduction Assay --- p.48 / Chapter 2.2.7.2 --- Assay of Plastic Adherence --- p.48 / Chapter 2.2.8 --- Flow Cytometric Analysis --- p.49 / Chapter 2.2.8.1 --- Surface Antigen Immunophenotyping --- p.49 / Chapter 2.2.8.2 --- Assay of Non-specific Esterase Activity --- p.50 / Chapter 2.2.8.3 --- Assay of Phagocytic Activity --- p.50 / Chapter 2.2.8.4 --- Assay of Endocytic Activity --- p.51 / Chapter 2.2.8.5 --- Cell Cycle/DNA Content Evaluation --- p.52 / Chapter 2.2.9 --- Gene Expression Analysis --- p.53 / Chapter 2.2.9.1 --- Cell Lysate Preparation --- p.53 / Chapter 2.2.9.2 --- Total RNA Isolation by cesium chloride isopycnic gradient --- p.53 / Chapter 2.2.9.3 --- Reverse Transcription --- p.54 / Chapter 2.2.9.4 --- Polymerase Chain Reaction (PCR) --- p.55 / Chapter 2.2.9.5 --- Agarose Gel Electrophoresis --- p.56 / Chapter 2.2.9.6 --- DIG 3，End Labeling of Oligonucleotide Probes --- p.57 / Chapter 2.2.9.7 --- Dot Blot Hybridization --- p.57 / Chapter 2.2.9.8 --- DIG Chemiluminescent Detection --- p.58 / Chapter 2.2.10 --- DNA Fragmentation Analysis --- p.59 / Chapter 2.2.11 --- Statistical Analysis --- p.60 / Chapter CHAPTER 3: --- EFFECTS OF FLAVONOIDS ON THE PROLIFERATION AND APOPTOSIS OF MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Results --- p.63 / Chapter 3.2.1 --- Growth-Inhibitory Effects of Flavone on Murine Myeloid Leukemia JCS Cells --- p.63 / Chapter 3.2.2 --- Cytotoxic Effects of Flavone on Murine Lymphocytes and Myeloid Leukemia JCS Cells --- p.67 / Chapter 3.2.3 --- Effects of Different Flavonoids on the Proliferation of Leukemia JCS Cells --- p.70 / Chapter 3.2.4 --- Anti-proliferative Effect of Flavonoids on Different Tumor Cell Lines --- p.74 / Chapter 3.2.5 --- Effects of Flavone and Flavonol on the Cell Cycle Kinetics of JCS Cells --- p.86 / Chapter 3.2.6 --- Induction of DNA Fragmentation of JCS cells by Flavone --- p.89 / Chapter 3.2.7 --- Effect of Flavone on the Clonogenicity of JCS Cells In Vitro and Tumorigenicity In Vivo --- p.92 / Chapter 3.3 --- Discussion --- p.94 / Chapter CHAPTER 4: --- EFFECTS OF FLAVONOIDS ON THE DIFFERENTIATION OF MURINE MYELOID LEUKEMIA JCS CELLS / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.2 --- Results --- p.100 / Chapter 4.2.1 --- Morphological Changes in Flavonoid-Treated JCS Cells --- p.100 / Chapter 4.2.2 --- Induction of Plastic Adherence in Flavonoid- Treated JCS Cells --- p.106 / Chapter 4.2.3 --- Surface Antigen Immunophenotyping of Differentiating JCS Cells --- p.106 / Chapter 4.2.4 --- NBT-Reducing Activity of Flavonoid-Treated JCS Cells --- p.114 / Chapter 4.2.5 --- Non-specific Esterase Activity of Flavonoid- Treated JCS Cells --- p.115 / Chapter 4.2.6 --- Endocytic Activity of Flavonoid-Treated JCS Cells --- p.116 / Chapter 4.2.7 --- Phagocytic Activity of Flavonoid-Treated JCS Cells --- p.117 / Chapter 4.3 --- Discussion --- p.118 / Chapter CHAPTER 5: --- MECHANISTIC STUDIES ON THE ANTI- PROLIFERATIVE AND DIFFERENTIAION-INDUCING ACTIVITIES OF FLAVONE ON MURINE MYELOID LEUKEMIA JCS CELLS / Chapter 5.1 --- Introduction --- p.122 / Chapter 5.2 --- Results --- p.125 / Chapter 5.2.1 --- Combinations of Flavone with Physiological Differentiation Inducers on the Proliferation and Differentiation of JCS Cells --- p.125 / Chapter 5.2.1.1 --- Modulatory Effects of Flavone and All-Trans Retinoic Acid (ATRA) on the Proliferation and Differentiation of JCS Cells --- p.125 / Chapter 5.2.1.2 --- "Modulatory Effects of Flavone and 1,25- dihydroxyvitamin D3 on the Proliferation and Differentiation of JCS Cells" --- p.130 / Chapter 5.2.2 --- Combinations of Flavone and Cytokines on the Proliferation and Differentiation of JCS Cells --- p.134 / Chapter 5.2.2.1 --- Modulatory Effects of Flavone and rmlFN-γ on the Proliferation and Differentiation of JCS Cells --- p.134 / Chapter 5.2.2.2 --- Synergistic Effects of Flavone and rmIL-1 on the Proliferation and Differentiation of JCS Cells --- p.137 / Chapter 5.2.3 --- Modulation of Cytokine Gene Expressionin Flavone-Treated JCS Cells --- p.144 / Chapter 5.3 --- Discussion --- p.159 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.165 / REFERENCES --- p.171

The grape flavonoids include anthocyanins, tannins and flavonols, all of which contribute to grape and wine quality by influencing the colour and mouthfeel of red wine. These compounds are synthesized in different parts of the berry and during different stages of berry development. In addition, environmental and viticultural factors such as light exposure can also alter the flavonoid composition of grapes. An understanding of how synthesis of these compounds is coordinated, their relationship to wine quality and the influence of bunch light exposure on the flavonoid composition of grapes, could be used to improve fruit quality by enhanced viticultural management. The first part of this study sought to investigate the relationship between the different products of the flavonoid biosynthetic pathway (anthocyanins, flavonols and tannins), from two climatic regions (warm and cool) and determine their role in grape and wine quality. In collaboration with a major winery, whole Shiraz grapes were sampled at the weighbridge from a range of different vineyards from two climatic regions; warm (Riverland) and cool (McLaren Vale) in 2003 and 2004. A total of 80 grape samples were collected in each season and processed (i.e. 100 berries, separated into skin, seeds and juice, weighed and frozen). Anthocyanins and flavonols were measured, in triplicate, in skins by HPLC. Tannins were determined in the skins and seeds by two methods; phloroglucinol hydrolysis (HPLC) and protein precipitation (UV-VIS spectrophotometer). A comprehensive comparison of the two methods is discussed. In both years, the grapes from warm and cool climates formed two distinct data sets based on flavonoid composition. There was a correlation between anthocyanins and flavonols for both the warm and cool climate samples in both years, however those from the warm region had lower anthocyanin for a given level of flavonol. As expected, the level of tannin in the seeds was greater than in skin for all samples. In both years, there was a weak correlation between anthocyanin levels in the skin and skin tannins, but no relationship with seed tannins. These results suggest there is some co-ordination in the synthesis of anthocyanins, flavonols and skin tannins. Also, the two regions clearly separated based on yield and despite the weak correlations in both regions, the levels of total anthocyanins were inversely related to yield. In addition, there was no relationship with any of the flavonoids and grape quality, indicating the need for improvement in streaming fruit for quality using these flavonoid compounds. The second part of the study was to investigate the effect of bunch light exposure on flavonol synthesis and accumulation in Shiraz and Chardonnay grapes during development. Light-excluding boxes were applied to bunches at budburst. Boxes were removed at four sampling times; flowering, pre-veraison, veraison and harvest. At each sampling time, berry skins were sampled when the boxes were removed and then every second day (light induced), along with exposed controls for one week. Flavonol accumulation and flavonol synthase (VvFLS1) gene expression was determined by HPLC and Real Time-PCR (RT-PCR) respectively. As expected, for all four sampling times, flavonol accumulation and VvFLS1 expression in the boxed fruit was significantly less than bunches exposed to light. On removal of boxes at flowering, pre-veraison and veraison, flavonols accumulated to levels similar to that of the exposed control fruit over a period of 4-6 days. There was a significant increase in VvFLS1 expression 2 days after exposure to light in parallel with the accumulation of flavonols. At harvest, in Chardonnay, VvFLS1 expression peaked by day 4, while in Shiraz VvFLS1 expression increased linearly and was highest at day 6. In contrast to the results for the earlier sampling times, the total amount of flavonols accumulated at harvest was less than 50% of exposed controls in Chardonnay and Shiraz grapes. These results show that flavonols are able to be induced by bunch light exposure at different times during berry development, including times when flavonols are not normally being synthesised. This suggests bunch light exposure can override the developmental control of flavonol accumulation. To further investigate the light induced expression of VvFLS1 in grapevines the molecular mechanism of transcriptional control was explored. Using genomic walking PCR techniques, two Shiraz VvFLS1 promoter sequences were cloned and their sequences were analysed. These promoter sequences were ~800bp in length and were 99% identical. A putative MYB responsive element (MRE) and several light responsive elements (LRE) were identified in the promoter region of these genes. To functionally test the VvFLS1 promoter(s), a transient assay was developed in Chardonnay suspension cells. Cells were bombarded with constructs containing potential transcription factors and the VvFLS1 promoter(s), fused to a luciferase reporter vector. After 48hrs incubation in the dark, cells were harvested and luciferase activity measured as an indicator of VvFLS1 promoter activity. Of the different transcription factors tested with the VvFLS1 promoter(s) the highest luciferase activity was observed using AtMYB12 (a flavonol-specific regulator of AtFLS1 in Arabidopsis (Mehrtens et al. 2005). While this result shows activation of the VvFLS1 promoters by AtMYB12 and the development of a transient reporter assay for testing the VvFLS1 promoter(s) a grapevine transcription factor specific for VvFLS1 was sought. Two techniques were employed to identify potential transcription factor regulators of the VvFLS1 promoter(s). The first involved BLAST sequence search analysis in a grapevine expression (EST) database with AtMYB12 and the second involved using DNA microarray technology to identify candidate transcription factors that were up-regulated in light exposed Chardonnay cell suspension cultures. Thirteen potential transcription factors were identified and after correlative RT-PCR analysis (with VvFLS1 expression patterns) two candidates were selected for further isolation and characterisation. These results have made significant progress in unravelling the molecular mechanisms of regulation of the flavonol biosynthetic, however additional experiments are required to unravel the transcriptional control of flavonol biosynthesis. This investigation contributes to our knowledge of flavonoid synthesis in grapes; how it is coordinated, the relationship with wine quality, and the influence of light particularly on synthesis of flavonols. It also explores the molecular mechanisms of VvFLS1 control, through isolation of the VvFLS1 promoter and identification of potential transcription factors, which may regulate it. An understanding of the synthesis of flavonoids and how they may be coordinated, particularly in response to light, could be used to improve fruit quality by enhanced viticultural management. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326767 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

The grape flavonoids include anthocyanins, tannins and flavonols, all of which contribute to grape and wine quality by influencing the colour and mouthfeel of red wine. These compounds are synthesized in different parts of the berry and during different stages of berry development. In addition, environmental and viticultural factors such as light exposure can also alter the flavonoid composition of grapes. An understanding of how synthesis of these compounds is coordinated, their relationship to wine quality and the influence of bunch light exposure on the flavonoid composition of grapes, could be used to improve fruit quality by enhanced viticultural management. The first part of this study sought to investigate the relationship between the different products of the flavonoid biosynthetic pathway (anthocyanins, flavonols and tannins), from two climatic regions (warm and cool) and determine their role in grape and wine quality. In collaboration with a major winery, whole Shiraz grapes were sampled at the weighbridge from a range of different vineyards from two climatic regions; warm (Riverland) and cool (McLaren Vale) in 2003 and 2004. A total of 80 grape samples were collected in each season and processed (i.e. 100 berries, separated into skin, seeds and juice, weighed and frozen). Anthocyanins and flavonols were measured, in triplicate, in skins by HPLC. Tannins were determined in the skins and seeds by two methods; phloroglucinol hydrolysis (HPLC) and protein precipitation (UV-VIS spectrophotometer). A comprehensive comparison of the two methods is discussed. In both years, the grapes from warm and cool climates formed two distinct data sets based on flavonoid composition. There was a correlation between anthocyanins and flavonols for both the warm and cool climate samples in both years, however those from the warm region had lower anthocyanin for a given level of flavonol. As expected, the level of tannin in the seeds was greater than in skin for all samples. In both years, there was a weak correlation between anthocyanin levels in the skin and skin tannins, but no relationship with seed tannins. These results suggest there is some co-ordination in the synthesis of anthocyanins, flavonols and skin tannins. Also, the two regions clearly separated based on yield and despite the weak correlations in both regions, the levels of total anthocyanins were inversely related to yield. In addition, there was no relationship with any of the flavonoids and grape quality, indicating the need for improvement in streaming fruit for quality using these flavonoid compounds. The second part of the study was to investigate the effect of bunch light exposure on flavonol synthesis and accumulation in Shiraz and Chardonnay grapes during development. Light-excluding boxes were applied to bunches at budburst. Boxes were removed at four sampling times; flowering, pre-veraison, veraison and harvest. At each sampling time, berry skins were sampled when the boxes were removed and then every second day (light induced), along with exposed controls for one week. Flavonol accumulation and flavonol synthase (VvFLS1) gene expression was determined by HPLC and Real Time-PCR (RT-PCR) respectively. As expected, for all four sampling times, flavonol accumulation and VvFLS1 expression in the boxed fruit was significantly less than bunches exposed to light. On removal of boxes at flowering, pre-veraison and veraison, flavonols accumulated to levels similar to that of the exposed control fruit over a period of 4-6 days. There was a significant increase in VvFLS1 expression 2 days after exposure to light in parallel with the accumulation of flavonols. At harvest, in Chardonnay, VvFLS1 expression peaked by day 4, while in Shiraz VvFLS1 expression increased linearly and was highest at day 6. In contrast to the results for the earlier sampling times, the total amount of flavonols accumulated at harvest was less than 50% of exposed controls in Chardonnay and Shiraz grapes. These results show that flavonols are able to be induced by bunch light exposure at different times during berry development, including times when flavonols are not normally being synthesised. This suggests bunch light exposure can override the developmental control of flavonol accumulation. To further investigate the light induced expression of VvFLS1 in grapevines the molecular mechanism of transcriptional control was explored. Using genomic walking PCR techniques, two Shiraz VvFLS1 promoter sequences were cloned and their sequences were analysed. These promoter sequences were ~800bp in length and were 99% identical. A putative MYB responsive element (MRE) and several light responsive elements (LRE) were identified in the promoter region of these genes. To functionally test the VvFLS1 promoter(s), a transient assay was developed in Chardonnay suspension cells. Cells were bombarded with constructs containing potential transcription factors and the VvFLS1 promoter(s), fused to a luciferase reporter vector. After 48hrs incubation in the dark, cells were harvested and luciferase activity measured as an indicator of VvFLS1 promoter activity. Of the different transcription factors tested with the VvFLS1 promoter(s) the highest luciferase activity was observed using AtMYB12 (a flavonol-specific regulator of AtFLS1 in Arabidopsis (Mehrtens et al. 2005). While this result shows activation of the VvFLS1 promoters by AtMYB12 and the development of a transient reporter assay for testing the VvFLS1 promoter(s) a grapevine transcription factor specific for VvFLS1 was sought. Two techniques were employed to identify potential transcription factor regulators of the VvFLS1 promoter(s). The first involved BLAST sequence search analysis in a grapevine expression (EST) database with AtMYB12 and the second involved using DNA microarray technology to identify candidate transcription factors that were up-regulated in light exposed Chardonnay cell suspension cultures. Thirteen potential transcription factors were identified and after correlative RT-PCR analysis (with VvFLS1 expression patterns) two candidates were selected for further isolation and characterisation. These results have made significant progress in unravelling the molecular mechanisms of regulation of the flavonol biosynthetic, however additional experiments are required to unravel the transcriptional control of flavonol biosynthesis. This investigation contributes to our knowledge of flavonoid synthesis in grapes; how it is coordinated, the relationship with wine quality, and the influence of light particularly on synthesis of flavonols. It also explores the molecular mechanisms of VvFLS1 control, through isolation of the VvFLS1 promoter and identification of potential transcription factors, which may regulate it. An understanding of the synthesis of flavonoids and how they may be coordinated, particularly in response to light, could be used to improve fruit quality by enhanced viticultural management. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326767 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Strawberries are a rich source of polyphenols which contribute to berry color and plant disease resistance, and have been shown to lower the risk of many chronic when consumed. While a considerable body of work exists on the polyphenolic composition of commercial strawberry (Fragaria x ananassa Duch.), less information is available concerning polyphenols in Fragaria vesca, or Alpine strawberry, considered a model system for the Rosaceae family of crop species. The study of natural and genetically-engineered F. vesca mutants with white fruit can provide unique insight into regulation of metabolic flux through the complex branched phenylpropanoid/flavonoid biosynthetic pathway. Thus, the identity and quantity of major phenolic-derived anthocyanins, flavonols, flavan-3-ols, hydroxycinnamic acids, and ellagic acid (EA)-derived compounds, of red-fruited versus white-fruited genotypes of F. vesca and F. x ananassa were compared by high performance liquid chromatography-mass spectrometry. Due to the unknown origin of all but one white-fruited mutant of F. vesca, it was assumed that each resulted from independent mutation events and would exhibit different flavonoid profiles. A total of 27 phenolic-derived compounds were identified. The white genotypes of both species had very low anthocyanin levels. Total content of free EA and its conjugated forms were generally higher in white than in red F. vesca, but were the opposite in F. x ananassa, more in red than in white berries. Differences in content of individual flavonoids and in group totals among the white F. vesca genotypes suggested that they may represent different mutations affecting flavonoid production. Polyphenol profiles of a red and a white cultivar of F. vesca during four fruit developmental stages were determined along with transcriptional analyses of key structural and regulatory genes of the phenylpropanoid/ flavonoid biosynthesis. The final concentration of polyphenolic groups in red versus white F. vesca was due to the differential expression patterns of key pathway genes, especially dihydroflavonol-4-reductase, anthocyanidin synthase, and UDP-glucose-flavonoid-3-O-glucosyltransferase. The efficacy of phenolic compounds were evaluated in an in vitro study for inhibiting growth of Colletotrichum spp. associated with anthracnose fruit rot of strawberry. Only trans-cinnamic, p-coumaric, and ferulic acid inhibited isolates of the pathogen.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Elderberry is very popular plant, that is due to its beneficial effects to human organism very reputable. The main content substances are flavonoids and anthocyanins. Fruits of this flower are available only seasonally (which is usual for most of flowers), so we have to search for alternative solution of getting them. One of these solutions is conservation. Nowadays there are lots of types of postharvest adjustment, so we have more opportunities to find an optimal concept for every one specific plant. The diploma thesis is focused on assessment of content of phenolic substances harvested in different parts of Hradec Králové. They were conservated at laboratory temperature, elevated (40řC, 60řC) and reduced (-18řC) temperature. Next part of thesis is focused on assessing of the content of anthocyanins in fruits, that had been conservated for 3 years in refrigerator. The theme was finding the best concept of postharvestal adjustment. Despite to the individual places of harvest, there were not mentionable differences found out in content of phenolic substances in fruits. Main differences in content of substances were caused by different temperature of conservating area. With the upper temperature the content of phenolic substances was lower. Storage in the fridge for 3 years did not have any influence...

62 10 ABSTRACT The object of this study was the influence of abiotic elicitor on the production of rutin in suspension culture of Fagopyrum esculentum Moench. The culture was cultivated in Murashigeho and Skoog nutritive medium with growth regulator: 2,4−dichlorfenoxyacetic acid (1 ml/l). The ultrasound was used as abiotic elicitor (0,1 W/cm3 , 35 kHz) for time period of 1, 2, 3, 4 and 5 min. The samples were taken 0, 6, 12, 24, 48, 72 and 168 hours after elicitation. The kontrol samples (without the influence of ultrasound) were taken 24 and 168 hours after elicitation. The amount of rutin was analyzed by HPLC. Suspension culture of Fagopyrum esculentum Moench. didn't produce any rutin under the influence of ultrasound. No release of rutin into the nutritive medium was observed during this study.

Currently, an increasing attention is being paid to phytochemicals as one of the most widely used chemopreventive compounds, generally considered as health-promoting and safe. Flavonoids representing a large group of phytochemicals are present in many dietary supplements formulated from natural sources. The consumption of these concentrated phytochemicals has dramatically increased in the recent decade. It appears, however, that the ingestion of flavonoids might be associated with some adverse effects. Some flavonoids are known modulators of enzymes involved in phase I and phase II metabolism of xenobiotics biotransformation, thus their induction may result in an increase of carcinogen activation. In this study, the effects of selected flavonoid compounds -naphthoflavone, - naphthoflavone, myricetin, and dihydromyricetin, and carcinogens (BaP, PhIP) on phase II metabolism enzymes, sulfotransferases (SULT), have been investigated. To determine the induction of SULT, antibodies for their immunodetection have been developed. Peptide antigens derived from sequences of selected rat sulfotransferases rSULT1A1, 1B1, 1C1, 1C2, 1C1/2, 1E1, and 2A1, were used as KLH conjugates for hen immunization to obtain yolk anti-peptide antibody (IgY). Fractions of IgY were isolated from eggs yolks by simple...

The aim of this work was to find out the content of flavonoids in the flowers of eleven cultivars of black elder (Sambucus nigra L.) and determine if their contents differ according to the statistical significance. Preparing the list of effects and substances that are contained in the flowers was the goal as well. The drug Sambuci nigrae flos is mostly used for its content of flavonoids and hydroxycinnamic acids in the therapy of colds and diseases of the urinary and respiratory tract. Its benefits were confirmed also by studies testing antibacterial and antiviral activity, diabetes and obesity, effects on the immune system and also protection against UV radiance. The content of flavonoids was established by the spectrophotometric method that is listed in the Czech Pharmacopoeia 2009 in the article Sambuci nigrae flos as the method for the determination of content. The statistic evaluation of the differences in the flavonoids content between the cultivars was made by ANOVA and the Bonferroni test. The demand of Czech Pharmacopoeia 2009 on the minimal content of flavonoids is 0,80%. This requirement was fulfilled in the flowers of these cultivars: Albida, Heidegg 13, Riese auß Voßloch, Sambu, Samdal, Sampo and Samyl. On the opposite side, flowers of the cultivars Allesö, Aurea, Dana and Juicy did...