| Natamycin is an efficient,safe and natural antifungal agent,which belongs to polyene macrocyclic antibiotic.However,its poor solubility and stability limit its application.In this present study,solubility and stability of natamycin were increased by cyclodextrin embedding technique.The solubility of various cyclodextrin derivatives were compared,and methyl-?-cyclodextrin(ME-P-CD)was choosen.The inclusion compound of natamycin and methyl-?-cyclodextrin was prepared by ultrasonic followed by freeze drying method.Complexation and thermodynamic constants were calculated to optimize the preparation conditions and the molecular binding mechism was identified by NMR,IR,molecular docking and molecular simulation.The effect of light on stability and antibacterial activity of inclusion compound was also investigated.Compared the effect for solubilization of natamycin with natural ?-cyclodextrins and its three hydrophilic derivatives:hydroxypropyl-?-cyclodextrin,methyl-p-cyclodextrin,maltose-p-cyclodextrin by phase solubility experiments.The results showed that methyl-?-cyclodextrin had the best solubilization effect and the highest complexing constant,indicating that methyl-?-cyclodextrin was the best wrapping material.The phase solubility experiment of different temperature showed that with the increase of temperature,the inclusion ability of methyl-?-cyclodextrin is poorer,and the complexing constant is lower,the thermodynamic constants can be seen through the calculation,and the inclusion process is entropy driven by spontaneous exothermic process.Through single factor experiment and response surface experiment,the optimum preparation conditions are as follows:ultrasonic time is 21 minutes,ultrasonic power is 59KW,and the molar ratio of methyl-?-cyclodextrin and azithromycin is 1.24:1.The model can predict the influence of various factors on the solubility of natamycin.Through the IR spectroscopy of natamycin,methyl-?-cyclodextrin,physical mixture and complex,we found that the primary amine ether ring molecular absorption peak of natamycin disappeared in the inclusion complex,and the number of absorption peaks appeared displacement.It indicated that the inclusion complex of natamycin and methyl-?-cyclodextrin was formed,and the large ring lactone part inserted into the hydrophobic cavity of cyclodextrin,and hydrogen bond force was formed between the carbohydrate of natamycin and methyl-?-cyclodextrin.Through 1HNMR experiments,the chemical shift of H3 and H5 in methyl-?-cyclodextrin were changed in complexes,even the chemical shift of H3 was maximal,and all most of the H in natamycin had a chemical shift.It indicated that inclusion complex have been formed,and the natamycin inserted from the wide side of methyl-?-cyclodextrin.The same results can be obtained from the 2D NOSEY NMR spectra.Combination of molecular docking and molecular dynamics simulation software,the combination of natamycin molecules with methyl-?-cyclodextrin molecule can be simulated and calculated,the results were consistent with the results of IR and NMR.In this paper,the stability of inclusion compound was studied by the method of bacteriostatic circle experiment and the kinetic model of degradation,it is found that cyclodextrin improved the stability of natamycin on the extreme pH(especially the strong acid)in the dark.Under UV irradiation,natamycin and inclusion follows a first-order degradation kinetics equation,fitting it and found that inclusion had smaller degradation coefficient and longer half-life under the UV light,it is indicated that the stability of natamycin under the UV light was improved because of the methyl-?-cyclodextrin.The results were similar under incandescent light irradiation.This conclusion in antibacterial test is also reflected,because natamycin degradated in ultraviolet light and incandescent light,so the antibacterial activity was greatly reduced,but the antibacterial activity of the inclusion was not affected.With the increase of solubility,the antibacterial activity also increased.In the UV lamps and incandescent light irradiation,the antibacterial activity of complexes unchanged,it is indicated that the inclusion does increase the Photostability of the natamycin. |
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