In order to clarify the molecular mechanism of the DNA damage and the intracellular target of Fr.3, UV�Cvisible absorption changes and a competitive assay employing EB were examined. The changes observed in the UV spectra may give evidence of the existing interaction mode. Generally, hyperchromism indicates that the complex binds to the negatively charged phosphate backbone at the periphery of the DNA, causing damage to the DNA GSK2118436 double helix. On the other hand, hypochromism and red shift indicate a conformational change of the DNA double helix. The changes observed in the UV spectra of the DNA after mixing it with Fr.3 indicated that Fr.3 might interact with DNA by the direct formation of a new complex with double helical DNA, causing double helix structural damage. The DNA double helix possesses many hydrogen bonding sites which are accessible both in the minor and major grooves, and it is possible that the components of Fr.3 might bond with DNA through hydrogen bonds, which in turn, may contribute to the hyperchromism observed in the absorption spectra. Competitive binding study with EB has been employed to study the interactions involved in DNA complex formation in order to investigate a potential intercalative binding mode. EB does not show any appreciable emission in buffer solution due to fluorescence quenching of the free EB by the solvent molecules. On addition of DNA, its fluorescence intensity is highly enhanced because of its strong intercalation between the adjacent DNA base pairs. Addition of a second molecule, which binds to DNA more strongly than EB, can decrease the DNA�Cinduced EB emission. The intensity of the emission band at 493 nm of the DNA�CEB system significantly decreased in C. michiganense subsp. sepedonicum genomic DNA, which indicated the competition of Fr.3 components with EB in binding to DNA. The quenching of DNA�CEB fluorescence suggested that Fr.3 components prevented EB from inserting into the DNA and Fr.3 could interact with DNA by intercalation. The cell cycle can be thought of as a circuit of regulatory components which, by SU5416 enabling an efficient flow of information, triggers events critical for cellular reproduction. The results indicated that the population of the treated cells at the I phase dropped to 66.68%, 65.39% and 62.51% respectively, compared with the control. We speculated that the components of Fr.3 inhibited RNA or protein which is related to cell division during the I phase. From Figure 13F, we know that the cell population at R phase increased to 33.32%, 35.64% and 37.49%, respectively, compared with the control. This indicated that Fr.3 disrupted R phase rather than I phase, causing most cells to remain in R phase. However, the results of the bactericidal kinetic assay revealed that inhibitory effect of Fr.3 occurred mainly during the logarithmic phase where the number of C. michiganense subsp. sepedonicum decreased significantly. These results indicated that Fr.3 led the cell population to arrest at R phase, with few cells passing through R phase into the cell division phase, finally resulting in a decrease in the number of C. michiganense subsp. sepedonicum. According to these results, we speculated that Fr.3 components disrupted the normal cell cycle of the bacteria, sequentially inhibiting the growth of the bacteria, and leading to cell lysis. Based on the present research, a schematic model of the proposed mechanism of Fr.3 is described in Figure 14. The active substances in Fr.3 resulted in loss of outer membrane integrity, causing outer membrane damage. The disruption of the cell membrane caused the leakage of cellular content, inhibition of the proton pump, respiratory chain, electron transfer and oxidative phosphorylation.