Study On The In Vitro Antifungal Activities And Actions Mechanism Of Furoquinoline Alkaloid Dictamnine

Clinical infectious diseases are usually caused by three main types of micro-organisms including viruses, bacteria and fungi, so the treatment of fungal infections is an important part of clinical anti-infective treatment. At present, the application of drugs against fungi are restricted, because the drugs have high toxicity and are easy to produce drug resistance resulting in the risng of the recurrence rate and re-infection rates. There was an urgent need to develop new antifungal drugs with high efficiency and low toxicity. Currently, it is an important research direction and hot spot in the search for antifungal active ingredient from Chinese herbal medicine. In the past century, people have found more than 300 kinds of Chinese herbal medicines with anti-fungal activity. Furoquinoline alkaloids dictamnine, isolated from the root bark of Dictamnus dasycarpus Turcz (Rutaceae plants dry root bark), which has the activities of inhibiting platelet aggregation, anti-insects, vascular relaxation and so on. Recent studies have shown that dictamnine can induce the cytotoxicity of cervical cancer, colon cancer and oral cancer cell in human. It is worth noting that dictamnine has obvious antifungi activity.At present, the combined application of antifungal agents has become one of the clinical important development of the clinical anti-fungal treatment. However, combination therapy at home and abroad reported are limited in a small number of combination between the antifungal agents and some combinations can produce the antagonism. So, screening the antifungal composition from Chinese herbal medicine and combining with current antifungal agents is an effective treatment program. Through the combination of antifungal agents can improve the therapeutic effect and reduce of the drug toxicity.This study investigated the in vitro drug sensitivity of dictamnine alone and in combination with fluconazole (FLC) against clinical isolates fungi and by using the tests of minimal inhibitory concentration recommended by CLSI and checkerboard microdilution method, agar diffusion test, time-kill curves. The results showed that dictamnine have the antifungal activity. The interaction between FLC and dictamnine was synergistic in 20 out of 26 Candida albicans strains, Trichophyton rubrum and Saccharomyces cerevisiae (S. cerevisiae). At the same time, the two drugs found no antagonism. FICIs (Fractional inhibitory concentration index) ranging from 0.25 to 1.5. The MIC value of two drugs used in combination was lower than that of each drug used alone, the result showed that fluconazole and dictamnine have significant synergy antifungal activity in vitro. The results of agar diffusion test show synergy more intuitively. When fluconazole at 16μg combinated with different dose of dictamnine, the inhibition zone expanded significantly compared to the control. The boundary was clear and there is no colony growth in inhibition circle. To our knowledge, this is first report on evaluation of in vitro antifungi activity against clinical pathgenic fungi in combination with fluconazole and dictamnine. MTT cytotoxicity experiment showed that high-dose of dictamnine (≥1024μg/mL)had the the toxic effects on spleen cells of health mice, and this dose of dictamnine was hihger than MIC value of dictamnine against clinical drug-resistant fungal. So, viewing from the cytotoxic, dictamnine can be used as lead compounds for drug design. The experimental results can provide a reference of clinical medicine with dictamnine.DNA microarray have a great value in drug screening, target gene identification, drug testing and administration of personalized delivery, gene discovery, genomic library mapping, species identification of traditional Chinese medicine, DNA research, computer applications, and so on. Fortune forecasted that bio-chip will has more impact on human than the micro-electronic chip in the 21st century. Gene chip provides a powerful tool in the study of gene function for the"post-genome project"and in modern medical science and medical diagnostics.The yeast S. cerevisiae is an ideal organism for the study of antifungal action, because S. cerevisiae has strong ability of adapting to environmental changes, which has similar characteristics to higher organisms, most notably because the genome sequence of S. cerevisiae has already been completed and the function of almost 70% of the genes is known. Additional, S. cerevisiae DNA chips commercialized can be used to indicate putative gene function, understand biochemical pathways, and investigate relationships between regulators and their target genes. The advantages mentioned above make the possibility of studying the mechanism of antifungal agents on whole-genome by using S. cerevisiae as a model. In this study, the chips will provide a powerful tool for studying the global gene expression profile of S. cerevisiae induced by dictamnine. Microarray data analysis revealed that a large number of genes (889) were differentially expressed in response to dictamnine treatment. 511 genes increased in expression and 378 genes were inhibited. Transcriptome data was interpreted using the hierarchical cluster tool, T-profiler. The genes included in multidrug resistance response, lipid biosynthesis pathway, sulfate assimilation, DNA replication and DNA recombination were significantly regulated by dictamnine. The transcription factors rRPE, PAC, Novel Filamenta, PDR3, MSN2/4, RPN4, TBP and UPC2 were also affected by dictamnine. The results of microarray can lay the foundation for the study on the mechanism of dictamnine against other pathogenic fungi, especailly yeast-like fungi.In addition, quantitative real-time RT-PCR and western-blotting were performed to verify the microarray results of selected genes. There was positive correlation between microarray data and real time RT-PCR data for all 11 genes, six genes were induced and five genes were reduced in response to dictamnine. The western-blotting results showed that the quantity of protein multidrug efflux protein Pdr5p was increased, which was consistent with the results of microarray. The results lays the foundation for further study on drug-resistance mechanisms of pathogenic fungi treated by dictamnine.In order to furtherly verify the microarray results and clarify the action mechanism of dictamnine against fungi, we studied the effect of dictamnine on model fungus S. cerevisiae using the methods of glucose-dependent efflux of rhodamine 6G, laser scanning confocal microscopy and flow cytometry. The results showed that dictamnine can inhibit the growth of S. cerevisiae, the higher drug concentration, the stronger inhibition. Large dosage of dictamnine (2×MIC) can effect the intra-cellular hereditary substance, the contents of DNA was increased and inhibit the budding and sporing of S. cerevisiae; dictamnine can activate the efflux pump of S. cerevisiae, this is consistent with the result of microarray and western-blotting; the results of flow cytometry showed that dictamnine can fracture the cell membrane. Tests mentioned above are to lay the foundation in further explore the mechanism of the active ingredient from anti-fungal medicine.This paper provides the new treatment protocols to clinical treatment of fungal infections; provides a useful theoretical basis for studying the action mechanisms of dictamnine against pathogenic fungi; and lays the foundation for the development and utilization of dictamnine.