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Pan, Xiaobei (2011) Anti-leukaemia and anti-bacterial activity of Evodia Rutaecarpa. Doctoral thesis, London Metropolitan University.
DNA topoisomerases are nuclear enzymes that maintain the appropriate level of DNA supercoiling and remove knots and tangles during many nuclear processes. These enzymes modulate the topological structures of DNA by transiently breaking DNA strand and rejoining the break. Topoisomerases are important targets for several anti-cancer and anti-bacterial drugs currently in clinical use. Many of these topoisomerase-targeted drugs, such as camptothecin and its derivatives, amsacrine and quinolone antibiotics, were found to structural contain a quinoline ring. The aims of this work are to isolate and characterize the quinoline alkaloids from Evodia rutaecarpa, evaluate the bioactivities of these quinoline alkaloids against both cancer cell lines and bacteria, and study their inhibitory activities toward topoisomerases. Chapter one presents a short review of topoisomerases including classification, mechanisms and their cellular functions, clinically used inhibitors of topoisomerase and quinoline alkaloids which possess structural similarity with several known topoisomerase inhibitors.
In chapter two, twenty peaks from Evodia rutaecarpa ethyl acetate crude extract (ECA) were separated by HPLC and fourteen of them were tentatively characterized by HR-ESI-MS/MS spectrometry, which include four quinazolinocarboline and ten quinolone alkaloids. However, only one new compound, furan-rutaecarpine, has not been previously reported in this plant. Eight compounds were collected, and were fully characterized unambiguously by 1H-, 13C- and 2D- NMR spectroscopy. Furthermore, the amount of two quinazolinocarboline alkaloids, evodiamine (10.38 mg/g in ECA and 0.24 mg/g in dry herb) and rutaecarpine (38.98 mg/g in ECA and 0.89 mg/g in dry herb), were determined by HPTLC.
Chapter three describes the anti-proliferative activities of ECA and the eight alkaloids isolated from the ext_ract against human leukaemia cell lines. The inhibitory mechanisms of these eight alkaloids on Topoisomerase I and II (Topo 1 and 11) were investigated. ECA exhibited anti-proliferative activities against two human leukaemia cells, K562 and THP-1 cells with IC50 values of 0.07 mg/ml and 0.13 mg/ml respectively, Among all the isolated alkaloids, evodiamine showed most effective inhibitory activity against the growth of human leukaemia cell lines. The.ICso values of evodiamine against human leukaemia cell lines, K562, THP-1, CCRF-CEM and CCRF-CEM/C1 (a camptothecin resistant cell line) were 34.35, 90.87, 4.70 and 2.92 µM, respectively. It also inhibited the hTopo I DNA relaxation activity with an IC50 value of 60.74 and hTopo II DNA decatenation activity with an IC50 value of 78.81 µM. The mechanistic studies demonstrated that evodiamine failed to trap the cleavage complexes of either hTopo I or II; was unable to bind or intercalate DNA; arrested cells in the G2/M phase of the cell cycle; and did not induce any detectable DN_A damage in cells. The results suggested evodiamine is a dual catalytic topoisomerase inhibitor. Moreover, the quinolone alkaloid 1-methyl-2- [(6Z,9Z)]-pentadecadienyl-4(1H)-quinolone at 100 µM inhibited 75.9% DNA relaxation activity of hTopo I and 85.7% DNA decatenation activity of hTopo II.
The anti-bacterial activities of the isolated quinoline alkaloids are presented in chapter four. The quinolone alkaloids with an alkyl group at the 2-position exhibited inhibitory abilities on the Gram-positive bacteria Staphylococcus epidermidis and Micrococcus luteus (MIC 128 µg/ml -1 µg/ml). Furthermore, the abilities of these quinolone alkaloids to inhibit the bacterial topoisomerases (DNA gyrase and Topo IV) were investigated. Two quinolone alkaloids with an alkenyl group at the 2-position inhibited- bacterial Topo II. 1-methyl-2-[(6Z,9Z)]-pentadecadienyl-4(1H)-quinolone inhibited both gyrase from Escherichia coli (IC50 142.55 µM) and Staphyllococcus aureus (IC50 35.21 µM). 1-methyl-2-[(Z)-5-undecenyl]-4(1H)-quinolone showed inhibitory activity against only E.coli gyrase (IC50 162.04 µM), and S. aureus Topo IV (IC50 87.26 µM).
In this study, fourteen alkaloids were isolated from Evodia rutaecarpa, eight of which were assayed for inhibitory activity against both Topo I and II. Evodiamine was the most active compound and was identified as a dual catalytic topoisomerase inhibitor. However, the exact step at which evodiamine inhibits the topoisomerase catalytic reaction is not known. Chapter five presents suggestions for investigation of its mode of action. The eight alkaloids were also tested for anti-bacterial activity and for inhibitory activity against DNA gyrase and Topo IV. Interestingly, the alkaloids which inhibited bacterial growth were inactive against bacterial Topo II. This indicated that there may be other cellular targets for these alkaloids and suggestions for future work in this area are included in chapter five. This study has demonstrated that the ethyl acetate extract of Evodia rutaecarpa contains alkaloids which have inhibitory activity against Topo I and II, or anti-bacterial activity and further investigation of bioactive compounds from this source is warranted.
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