Synthesis, Antibacterial Activities And Its Mechanism Of Polythionate Antibiotics

To date, few investigations have focused on the antibacterial and antifungal properties of polythionates, as intermediates in the oxidation process of inorganic sulfur compounds, due to their synthetic difficulty and commercial unavailability. Here, we developed a new approach for preparing high purity potassium hexathionate and potassium pentathionate through interface reaction from sodium thiosulfate and sulfur chloride(sulfur dichloride for the synthesis of pentathionate). Potassium tetrathionate and potassium trithionate were prepared with modifications of the sulfur chloride(sulfur dichloride for the synthesis of trithionate) /potassium pyrosulfite method. The purity of these polyhionates was determined by capillary electrophoresis and by titration with Hg Cl2 and found to be 99.8%(titration, 98.81%), 99.7%(titration, 98.47%), 99.91%(titration, 99.08%), and 99.95%(titration, 98.54%), respectively.The antibacterial activities of potassium polythionates against Gram-positive Staphylococcus aureus(S. aureus, ATCC 35696) and Gram-negative Pseudomonas aeruginosa(P. aeruginosa, ATCC 27853) and Escherichia coli(E. coli, ATCC 23282) were determined by minimum inhibitory concentration(MIC), minimum bactericidal concentration(MBC) analyses, and cup diffusion methods. The antibacterial effect of potassium polythionates, from trithionate to hexathionate, improves with the increase in length of the sulfur chain of the polythionates, which primarily results from the gradually increasing sulfur-release of polythionates. Since the reaction between the Lewis base(e.g., urea, potassium carbonate) and sulfuric acid, which resulted from the hydrolysis of potassium polythionates, stimulates the hydrolytic reaction and results in the formation of more active sulfur, the application of polythionates with different bases(carbonate, urea) exhibits enhanced antibacterial activity compared to application of pure polythionates. The MICs of pure potassium hexathionate against P. aeruginosa and S. aureus were 1.95 and 125 μg.m L-1, respectively. The corresponding MBC value against P. aeruginosa was 1000 μg.m L-1. However, there is no bactericidal activity against S. aureus and no bacterial ability against E. coli in the concentration range tested. The MIC and MBC values of potassium hexathionate contained in the urea-hexathionate mixture, against P. aeruginosa, only have 0.98 μg.m L-1 and 250 μg.m L-1, respectively. Since urea has no bactericidal effect against the test strains in the concentration range tested, results evidence that the addition of urea to the solution of hexathionate improves the antibacterial and bactericidal abilities of hexathionate for about 2 and 4 times, respectively. And the antibacterial abilities of hexathionate against S. aureus was also found to be enhanced for 4 times. Similar results were appeared in the case of potassium pentathionate. The MIC and MBC values of urea-pentathionate mixture against P. aeruginosa are 7.81 μg.m L-1 and 500 μg.m L-1, respectively. However, the MIC value of pure potassium pentathionate against P. aeruginosa is 62.5 μg.m L-1. It is showed that the addition of urea to the solution of potassium pentathionate improves the antibacterial and bactericidal abilities of hexathionate for about 8 times.Like in the case of the addition of urea, the enhanced antibacterial activities were also observed in the solution of carbonate-polythionate mixture. Though potassium carbonate has no antibacterial effect against test strains in the concentration range tested, the MIC value of carbonate-hexathionate mixture against S. aureus is 7.81 μg.m L-1. The MIC and MBC values of carbonate-hexathionate mixture against P. aeruginosa are 0.98 and 250 μg.m L-1, respectively. Thus, results evidence that the addition of carbonate to the solution of hexathionate improves the antibacterial abilities of hexathionate, against P. aeruginosa and S. aureus, for about 2 and 16 times, respectively. Similar to the result of carbonate-hexathionate mixture, the MIC and MBC values of carbonate-pentathionate mixture against P. aeruginosa are 1.95 and 500 μg.m L-1, respectively. In contrast, corresponding MIC value of pure potassium pentathionate is 62.5 μg.m L-1, which further indicates that adding potassium carbonate to the solution of potassium pentathionate enhances its antibacterial abilities, against P. aeruginosa, for 32 times.The antifungal activities of potassium polythionates against F. moniliforme(F. moniliforme, Gene Bank:GQ466389), F. graminearum(F. graminearum, Gene Bank: GQ466391), and F. oxysporum f.sp.vasinfectum(F. oxysporum, Race-7) were measured by the spread plate method. Like in the case of its antibacterial ability, the gradually increasing antifungal activities of potassium polythionates were evidenced from tetrathionate to hexathionate in the concentration range tested. Though potassium hexathionate presents the strongest antifungal effect than other polythionates in the inoculation early, the antifungal activities gradually diminishs with age. For comparison, the antifungal effect of potassium pentathionate is about five times as that of potassium tetrathionate, and the basic antifungal stability of potassium pentathionate with age showed that potassium pentathionate is promising as a cheap and sustained antifungal candidate. The antifungal effect of all potassium polythionates, under the same conditions, is strengthened gradually from F. moniliforme, F. graminearum, to F. oxysporum. As an example, the antifungal activities of potassium pentathionate against F. moniliforme and F. graminearum is not observed when the concentration was less than 1000 μg.m L-1and 500 μg.m L-1, respectively. In contrast, increasing the concentration to 250 μg.m L-1 or more generated antifungal effect against F. oxysporum. For three fungus tested, however, the higher the concentration of all potassium polythionates, the stronger the antifungal ablity is.In order to improve the biological activities of potassium polythionates and modify their physical and chemical properties for being used in special fields, Herein, we proposed a convenient and green process to prepare hexadecyltrimethylammonium polythionates through metathesis reaction using the aqueous solutions of hexadecyltrimethylammonium chloride and homemade potassium polythionates. The structure and physical properties of hexadecyltrimethylammonium polythionates were characterized by 1H NMR, IR, MS, melting point, solubility and TG-DSC, respectively. Their antibacterial and antifungal properties were evaluated by cup diffusion method and inhibition rate, respectively. Results showed that hexadecyltrimethylammonium polythionates have stronger antibacterial and antifungal effect than that of their corresponding potassium polythionates due to the synergistic effect between quaternary ammonium cation and active sulfur species from the decomposition of polythionates.To analyze the antimicrobial mechanism of polythionates against bacteria and fungi, the change of fungal morphology and fungal components, before and after medication, was investigated by in situ optical microscopic observation and X-ray photoelectron emission microscopy, respectively. It was showed that the hypha of control F. oxysporum stretched fully, however, the reunion bending mycelia of F. oxysporum cultured by polythionates PDA was appeared, and the hypha and spore contain a plenty of violet material compared with the control fungal. Furthermore, the content of potassium and sulfur of F. oxysporum cultured by potassium polythionates PDA was higher than that of the control F. oxysporum. Similarly, the F. oxysporum cultured by hexadecyltrimethylammonium polythionates PDA contain more carbon and sulfur, which indicates that polythionates has poison effects of inner absorber.Finally, to investigate the technical feasibility of polythionates used as pesticide fertilizer, living plant experiments,based on laboratory experiments, of tomato and strawberry seedlings was carried out under solution culture. It was found that both potassium polythionates and hexadecyltrimethylammonium polythionates, with appropriate concentrations, can boost the growth of tomato and strawberry seedlings while protecting them from harmful fungal infection.