http://dspace.cityu.edu.hk:80/handle/2031/728 2013-04-30T09:58:54Z 2013-04-30T09:58:54Z Zhang, Qi ( 張琦) http://dspace.cityu.edu.hk:80/handle/2031/6585 2012-08-07T07:46:16Z 2010-01-01T00:00:00Z

Title: The biological activities and molecular mechanism of a herbal formula extract of Rehmanniae Radix and Astragali Radix in wound healing Authors: Zhang, Qi ( 張琦) Abstract: Wound healing is an intricate process that involves a variety of cell types, including fibroblast, platelet, neutrophil, macrophage, endothelial cell and keratinocyte, as well as a set of biochemical events. The classical model of wound healing is divided into four sequential but overlapping phases: hemostasis, inflammation, proliferation and remodeling. In pathological conditions, such as diabetes, an organized and orderly wound healing process is interrupted and results in chronic wounds. Fibroblast is the connective tissue cell. During the proliferation phase of wound healing, fibroblasts are stimulated by various growth factors to proliferate and migrate to the wound site, and produce extracellular matrix (ECM) including collagen and fibronectin. Fibroblast is one of the components making up granulation tissue in wound healing. It is also able to secrete growth factors associated with angiogenesis and epthelialization, such as vascular endothelial cell growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGFβ). A Chinese herbal formula, consisting of Rehmanniae Radix (RR) and Astragali Radix (AR), has been clinically demonstrated to improve the healing of diabetic foot ulcer in diabetic patients with insulin resistance. In vivo study has shown that RR extract reduced the wound area and promoted scar formation in a diabetic foot ulcer rat model. In the present study, the biological activities and molecular mechanism of the herbal formula were investigated for wound healing using the human foreskin fibroblast cell model. The formula was prepared based on the weight ratio of AR to RR at 2:1. Fibroblasts were exposed to water extract of the herbal formula (Formula AR-RR) in a serum-free medium. The results showed that Formula AR-RR at the concentration of 4mg/ml significantly increased cell viability and proliferation. Flow cytometry and Western blot analysis demonstrated that Formula AR-RR was able to affect fibroblast cell cycle, and stimulate cells to enter S phase with the expression of cyclin D1 protein. Fibroblast cell migration, adhesion and contraction were subsequently studied to further understand the effect of Formula AR-RR on wound healing. Formula AR-RR was found to stimulate fibroblast migration and to improve the adhesion of fibroblasts to the ECM protein, fibrinogen. Wound contraction occurs at a late stage of wound healing. The evaluation of fibroblast contraction revealed that Formula AR-RR did not significantly enhance cell contraction. Differentiation of fibroblasts into myofibroblasts is responsible for providing the force for wound contraction. Western blot demonstrated that the expression level of alpha-smooth muscle actin (α-SMA) protein, a cytoskeletal protein biomarker indicating the differentiation of fibroblast to myofibroblast, was not changed, suggesting that Formula AR-RR had no effect on the differentiation of fibroblast to myofibroblast and the promotion of cell contraction. Wound repair is dependent on the expression and deposition of ECM proteins, mostly collagen and fibronectin, in fibroblasts. Formula AR-RR was also found to elevate the expression of ECM proteins, including collagen I, III and fibronectin in fibroblasts. Because the deposition of ECM proteins is associated with collagenases responsible for the breakdown of the matrix protein, the cytoplasmic matrix metalloproteinase protein (MMP) and the gene expression of tissue matrix metalloproteinase inhibitor (TIMP) were measured in cells treated with Formula AR-RR. The results showed that the stimulation of fibroblasts by Formula AR-RR increased TIMP1 gene expression and decreased the synthesis of MMP3 protein. Many diverse biological events during wound healing, including cell growth, cell migration, angiogenesis, fibrogenesis, and inflammatory response, are driven by cytokines. In the present study, Formula AR-RR was found to induce fibroblasts to increase the synthesis of angiogenic and fibrogenic factors in the cytoplasm, including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), transforming growth factor beta 1 (TGFβ1) and bone morphogenic protein 6 (BMP6). The induced production of VEGF, PDGF, TGFβ1 and BMP6 suggested that Formula AR-RR was able to affect blood vessel formation and maturation, help form granulation tissue, and promote acute wound repair. In addition, Formual RA-RR decreased the production of inflammatory factors, including tumor necrosis factor alpha (TNFα), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), interleukin-6 (IL-6) and interleukin-8 (IL-8), which are responsible for recruiting neutrophils leading to a chronic wound. The gene expression profile of fibroblasts stimulated by Formula AR-RR was studied by using a cDNA microarray containing 10,000 human genes. One hundred and twenty-six genes with significant change of expression level were identified, with 102 genes up-regulated and 24 genes down-regulated. Further analysis identified two pathways involved in wound healing - a Wnt signaling pathway and an angiogenesis signaling pathway. Eighteen genes with significant differential regulation were classified in the Wnt signaling pathway. Proteins encoded by these genes were distributed in 11 components of this pathway, including frizzled (FZD1, 2.3-fold), casein kinase 1 (CSNK1E, 0.5-fold), cadherin (PCDH1, 4.2-fold; FAT, 3.7-fold; PCDHGC3, 2.0-fold; CDH16, 2.5-fold; CELSR3, 2.9-fold and PCDH8, 2.2-fold), protein kinase C (PRKCH, 3.0-fold and PRKCZ, 2.6-fold), protein phosphatase 2A (PPP2R5A, 2.4-fold), calcineurin (PPP3CB, 2.4-fold), B-cell lymphoma 9 (Bcl9, 8.6-fold), switched/sucrose non fermentation (SMARCC1, 4.7-fold and SMARCA5, 2.2-fold), transforming growth factor beta activated kinase 1 (ACVR1B, 2.4-fold), CREB binding protein (CREBBP, 13.4-fold), and Wnt target genes (CCND1, 2.3-fold). Proteins encoded by ten genes were identified to involve in eight components of the angiogenesis signaling pathway, including protein kinase C (PRKCH, 3.0-fold; PRKCZ, 2.6-fold), sphingosin kinase (SPHK1, 2.6-fold), mitogen activated protein kinase 4 (MAPK4, 2.6-fold), platelet-derived growth factor (PDGFA, 3.7-fold), growth factor receptor-bound protein 7 (GRB7, 2.3-fold), growth factor receptor-bound protein 14 (GRB14, 20.7-fold), frizzled (FZD1, 2.3-fold), and delta/serrate (DLL1, 6.0-fold and DLL4, 3.6-fold). These two pathways are directly related to cell proliferation, angiogenesis, and ECM formation in the process of wound healing, and provide the molecular basis for the effects of Formula AR-RR. In summary, the study showed that Formula AR-RR has obvious biological effects on wound healing through promoting fibroblast proliferation, increasing cell migration and adhesion, stimulating the synthesis of cytokines involved in angiogenesis and fibrogenesis, and controlling inflammation. Recently, some chemical components, such as formononetin and genistine, were identified from Formula AR-RR. Formononetin was reported to promote angiogenesis, and genistine was found to substantially impair inflammatory response. Identification of additional bioactive chemical components from Formula AR-RR, and analysis of their functions in the future will further help understand the mechanisms of the herbal formula and develop new therapeutics for wound healing. Notes: CityU Call Number: RM666.H33 Z36 2010; xvi, 215 leaves : ill. (some col.) 30 cm.; Thesis (Ph.D.)--City University of Hong Kong, 2010.; Includes bibliographical references (leaves 197-215)

2010-01-01T00:00:00Z Zeng, Lingbin ( 曾令斌) http://dspace.cityu.edu.hk:80/handle/2031/6584 2012-08-07T07:46:13Z 2011-01-01T00:00:00Z

Title: An investigation on the bioactive properties of the chemical components and endophytic fungi of Cephalotaxus oliveri Mast. Authors: Zeng, Lingbin ( 曾令斌) Abstract: It is widely accepted that endophytic fungi are a rich source of active compounds. Many natural products with diverse bioactivities have been obtained from these fungi, including ones, such as taxol and podophyllotoxin, which were originally thought to be isolated from plants. These results imply that endophytic fungi are a potentially large source of untapped bioactive compounds awaiting exploration. Cephalotaxus oliveri Mast. is a relic, dioecious plant species and endemic to some subtropical forests in China. Its richness in harringtonine (HT) and homoharringtonine (HHT), two phytochemical drugs used to treat leukemia and other cancers is well established. To date, little information is available on the bioactivities of the chemical components and the endophytic fungi of C. oliveri. Hence, this plant seems to be a good candidate to explore the bioactivities of its chemical components and endophytic fungi, to analyze the chemical profile and their antioxidant activities, to isolate and identify the endophytic fungi, and evaluate their bioactivity potential, and to search for HT- and/or HHT- producing strains amongst these fungi, and finally to isolate the pure active compounds from the endophytic strain with the most potent bioactivities. Antioxidant activities of C. oliveri as indicated by the DPPH (2,2-diphenyl -1-picryl hydrazyl), ABTS [2,2'-azinobis (3-ethyl- benzothiazoline-6- sulfonic acid)], superoxide, reducing power coupled with the total phenolic content assays suggest the plant has potent antioxidant potentials. The methanol extract had the most potent activities in DPPH, ABTS and reducing power assays with the highest level of phenolic content among the test extracts. LC-MS/MS and GC-MS analyses resulted in the detection of 22 compounds in the methanol extract, among which are 15 phenolic compounds. The considerable antioxidant potentials and high phenolic content suggest C. oliveri is a rich source of natural phenolic antioxidants. A total of 101 endophytic fungal strains were isolated from C. oliveri using successive surface-sterilization, fragment plating and hyphal tip methods. Nineteen representative strains, identified as either Guignardia (anamorph Phyllosticta) or Colletotrichum species by 18S rRNA gene sequencing, show evident antioxidant, anticancer and antimicrobial activities. The phenolic compounds produced by the endophytes contributed to their antioxidant activities as assessed in DPPH test. Among the endophytic strains, Guignardia sp. L79 was the most antioxidative and cytotoxic. LC-MS/MS and GC-MS analyses indicate the presence of four phenolic compounds including protocatechuic acid, gentisic acid, tyrosol and syringol as secondary metabolites in Guignardia sp. L79. High yields suggest a promising alternative source of the four phenolic compounds can be produced by controlled fungal fermentation. Tyrosol and syringol were isolated from the ethyl acetate extract of Guignardia sp. L79 culture liquid by bioactivity guide fractionation. Syringol displayed more potent antioxidant activities than the commonly used synthetic antioxidant BHT in DPPH, ABTS and reducing power assays, whereas tyrosol exhibited only weak activities in the three assays. Syringol had overt cytotoxic activities to all the three test cancer cell lines, while tyrosol exerted significant inhibitory action to KB and HeLa cells. This is the first report on tyrosol from Guignardia (anamorph Phyllosticta) species and syringol from endophytes as secondary metabolites. However, no trace of HT and HHT was detected in the culture filtrates of all the 101 endophytic strains from C. oliveri using HPLC-DAD analysis. Notes: CityU Call Number: QK494.5.C4 Z46 2011; xi, 191 leaves : ill. 30 cm.; Thesis (Ph.D.)--City University of Hong Kong, 2011.; Includes bibliographical references (leaves 144-176)

2011-01-01T00:00:00Z Yu, Lei ( 虞磊) http://dspace.cityu.edu.hk:80/handle/2031/6583 2012-08-07T07:46:11Z 2011-01-01T00:00:00Z

Title: A high efficient anaerobic bio-system for azo dye wastewater and the decolorization mechanism Authors: Yu, Lei ( 虞磊) Abstract: Azo dyes are a major class of synthetic dyes and are being extensively discharged in high concentrations in the effluents of textile, food, paper printing, and cosmetic industries. Water contamination by azo dyes is not only arousing esthetic problems but also is causing toxic impacts on aquatic lives and even threatening human health. Biological processes have been recognized as an inexpensive and environmentally friendly way to treat azo-dye-rich wastewaters. The main focus of this research is the use of methyl orange (MO) as a model dye to carry out a detailed study on the anaerobic biodegradation of azo dye. The biological decolorization of methyl orange (MO), a typical azo dye, was investigated in anaerobic sequenced batch reactors (ASBRs). These reactors were adapted to an MO loading rate of up to 1.5 g l-1 day-1 with complete dye decolorization achieved in each cycle. The anaerobic sludge was found to have a saturated MO adsorption capacity of 36 ± 1 mg g MLSS-1. UV/visible spectrophotometer and high-performance liquid chromatography analytical results indicated that the MO adsorption and decolorization occurred simultaneously. In light of the considerable adsorption ability of anaerobic sludge, a modified two-stage pseudo-first order kinetics that takes into account adsorption and decolorization were established to describe the MO removal process. Performance of MO decolorization under different substrate and salt (NaCl) concentrations were also studied. A high salt concentration was found to pose negative effects on MO decolorization. A Monod equation was introduced to describe the inhibition effects of salt on the biodegradation process, since a first-order kinetic was not able to reflect the inhibition type and level. The calculated parameter Rmax (maximum decolorization rate) decreased from 92.6 to 17.2 mg l-1 h-1, when salt concentration increased from 0 to 40 g l-1, while the Ks (half saturation concentration) value varied only slightly and non-systematically. The negative correlation between Rmax and salt concentration indicated that salt inhibition is of noncompetitive nature, and the inhibition constant KI was calculated to be 3.67 g-NaCl l-1. In order to offer a better insight into the decolorization mechanisms of azo dyes, pure microbial cultures have also been used to explore the MO decolorization process. Specifically, a facultative anaerobic bacteria strain GS-4-08 was isolated from an ASBR and used for MO decolorization. This bacterium strain was identified as a member of Klebsiella oxytoca based on Gram staining, morphology characterization and 16S rRNA genes analysis. It exhibited good ability in simultaneous azo dye decolorization and hydrogen production in the presence of an appropriate electron donor (ED). The optimum pH, temperature and ED type and concentration for MO decolorization were 7, 35 oC and 20 mM of sucrose, respectively. This strain was able to efficiently convert ED into H2 and other valuable products (e.g., ethanol and acetate) during MO decolorization even at a high MO concentration of 0.5 mM, indicating a high MO tolerability. Hence, the use of this bacterium strain for MO decolorization not only avoids the inhibition of accumulated volatile fatty acids (VFAs) during MO decolorization, but also enables recovery of energy at the same time. Furthermore, a possible co-metabolism mechanism of MO decolorization and H2 production by the genus GS-4-08 was proposed based on the analysis of the intermediate and end products. A membrane-associated pyruvate dehydrogenase is likely to be the primary enzyme responsible for NADH2 generation. The hydrogenase and some other dehydrogenases involved in this system are mainly for hydrogen production, while the membrane-bound putative azoreductase functions as the terminal reductase. NADH2 produced during glucose hydrolysis and the primary dehydrogenases that serve as electron donors are transported through the electron transport chain to the azoreductase, driving the cleavage of azo bonds. This possible mechanism of electron transfer explains why higher decolorization rate was obtained when sugars and pyruvate were used as substrates than when other end products of glycolysis (e.g., ethanol, acetate, formate and hydrogen) were utilized. Notes: CityU Call Number: TD756.45 .Y8 2011; xvi, 158 leaves : ill. 30 cm.; Thesis (Ph.D.)--City University of Hong Kong, 2011.; Includes bibliographical references.

2011-01-01T00:00:00Z Yan, Zhongzheng ( 閆中正) http://dspace.cityu.edu.hk:80/handle/2031/6581 2012-08-07T07:46:06Z 2011-01-01T00:00:00Z

Title: Responses and tolerance mechanisms of mangrove seedlings to heavy metal stress Authors: Yan, Zhongzheng ( 閆中正) Abstract: The strategies of higher plants to cope with heavy metal stresses vary with stress types and durations, as well as plant species. The present research aims to (1) examine the effects of different levels of lead (Pb) stress on seedlings of Avicennia marina and compare the growth and biochemical responses to Pb stress between seedlings with experimentally removed cotyledons prior to their natural fall off and those with cotyledons; (2) investigate the temporal responses of oxidative enzymes activities, compatible osmolytes and polyphenols in seedlings of three common mangroves plants, Kandelia obovata, Excoecaria agallocha and Acanthus ilicifolius under Pb or manganese (Mn) stresses and (3) compare the responses of three endogenous phytohormones, namely jasmonic acid (JA) and salicylic acid (SA), in leaves of K. obovata, E. agallocha and A. ilicifolius to Pb or Mn stresses. The effects of Pb (0-1000 mg L-1) stress on the growth and biochemical responses of seedlings of A. marina, with and without cotyledons, were examined. After 50 days exposure to Pb, the growth of A. marina was not affected at low Pb stress (0-250 mg L-1). Roots could tolerate high Pb stress and a significant reduction in biomass was only found at the highest Pb level (1000 mg L-1). Pb was mainly accumulated in roots, with some uptake in cotyledons but very little in leaves. Pb stress level had significant positive effects on sugar content, malonaldehyde (MDA) concentration and peroxidase (POD) activity in both roots and leaves, irrespective to the presence or absence of cotyledons. The removal of cotyledons only showed significant effects on POD activity and MDA content in roots. Sugar content in the cotyledon of Pb-treated seedlings was significantly lower than that in the control, suggesting that more carbohydrate reserves stored in cotyledons had been mobilized. The changes of growth, accumulation and translocation of heavy metals (Pb or Mn) in seedlings of K. obovata, E. agallocha and A. ilicifolius under different levels of Pb or Mn stresses were followed at different stress time. Pb or Mn stress had no significant effect on growth of mangrove seedlings at Days 1 and 7 but a significant decline in growth was observed at Day 49. Both Pb and Mn were accumulated in root tissues of the seedlings at levels higher than that in sediment. The amount of Mn accumulated in leaves was significantly higher than that in roots, as Mn is an essential plant micronutrient which has higher mobility than Pb. Among the three species, translocation of Pb from root to leaf was faster in E. agallocha and A. ilicifolius than in K. obovata, and significant accumulation of Pb was found in leaves of the first two species at Day 1. The temporal responses of antioxidative enzyme activities, including superoxidase dismutase (SOD) and POD; concentrations of phenolic compounds, total polyphenols (TP) and extractable condensed tannin (ECT) and compatible osmolyte, proline and soluble sugars in seedlings of K. obovata, E. agallocha and A. ilicifolius to Pb or Mn stresses were investigated. SOD and POD activities in all three species exhibited rapid responses to Pb or Mn stresses, with a decline of POD activity but an increase of SOD activity observed in both roots and leaves of K. obovata at Day 1. However, both POD and SOD in E. agallocha and A. ilicifolius increased significantly in roots and leaves at Day 1. With increases in stress time, at Day 7, POD and SOD in leaves of K. obovata, E. agallocha and A. ilicifolius significantly augmented at the moderated Pb or Mn levels. At Day 49, both SOD and POD activities in leaves of K. obovata were significantly reduced but were higher in roots than at Day 7, irrespective to the stress level. As compatible osmolytes, the changes of proline and soluble sugar content due to Pb or Mn stress were not as fast as that of the antioxidative enzymes. Significant increases of proline concentrations were detected in leaves of K. obovata, E. agallocha and A. ilicifolius at Days 7 and 49 but not at Day 1. Soluble sugars seemed less sensitive than proline in leaves of E. agallocha and A. ilicifolius, which only increased significantly at Day 49. The accumulation of soluble sugars in roots was more rapid than that in leaves, as Pb or Mn stresses induced significant increases of soluble sugars in roots of K. obovata, E. agallocha and A. ilicifolius at Day 7. These results showed that proline changes in leaves of mangrove seedlings were more rapid than soluble sugars under heavy metal stress, suggesting that proline was the primary compatible osmolytes adopted by mangrove seedlings in response to heavy metal stress. The changes of phenolic compounds under metal stress were also slower than that of the antioxidative enzymes. TP and ECT concentrations in both roots and leaves of K. obovata, E. agallocha and A. ilicifolius showed no significant changes at Day 1. As stress time increased, TP and ECT decreased significantly in roots with metal levels at Days 7 and 49. Mn-treated roots had significantly lower phenolic concentrations than the Pb-treated roots, suggesting that mangrove seedlings were more sensitive to Mn than Pb stress. In leaves of K. obovata, E. agallocha and A. ilicifolius, TP and ETC concentrations increased significantly at low levels of metal stress (100 and 200 mg Kg-1 Pb or Mn), implying that mangrove seedlings used the phenolic compounds as the defensive mechanism to metal stress. The quantitative analysis of the responses of two plant hormones, JA and SA to different levels of Pb or Mn stress in leaves and roots of K. obovata, E. agallocha and A. ilicifolius at different stress time were followed using gas chromatography-mass spectrometry. Mangroves seedlings, similar to other wetland plants, increased their endogenous SA and JA concentrations which contribute to their tolerance to heavy metal stress. Increases of SA and JA were mainly observed in leaves of K. obovata and E. agallocha, especially at Day 1, while such increase was only observed at Days 7 and/or 49 in A. ilicifolius. The present research revealed that different mangrove species showed different responses and tolerance mechanisms to heavy metal stresses at different stress time. K. obovata showed stronger tolerance abilities to Pb and Mn than E. agallocha and A. ilicifolius. The tolerance ability of mangrove seedlings attributed, in part, to the size of the propagule, and the energy reserves in the propagule may play important role in the anti-stress processes. When the mangrove seedlings were subject to heavy metal stress, the quickest responses were observed on the hormonal and antioxidative enzymes levels. Other mechanisms, such as the synthesis of osmolytes, proline and soluble sugar, and the synthesis of the polyphenols occurred at a later stage of stress. The tolerant species, like K. obovata and E. agallocha, tended to acclimate to metal stresses by increasing and maintaining high levels of SOD and POD activities at later stage of the treatment, while the non-tolerant species such as A. ilicifolius could not acclimate to the metal stress and enzyme activity decreased with the time. Notes: CityU Call Number: QK938.M27 Y36 2011; xxxiii, 274 leaves : ill. 30 cm.; Thesis (Ph.D.)--City University of Hong Kong, 2011.; Includes bibliographical references (leaves 253-273)

2011-01-01T00:00:00Z