Bioguided isolation, identification and bioactivity evaluation of anti-MRSA constituents from Morus alba Linn.

The root bark of Morus alba Linn. (M. alba), a traditional folk medicine, has been documented in the Chinese Pharmacopoeia, which has been widely used for asthma, fever, pneumonia, edema, vomit, colitis, bronchitis and keratitis diseases. Some of the diseases may be related to respiratory, digestive, urinary tract infections. Although Diels-Alder adducts (DAAs), flavonoids, 2-arylbenzofurans and stilbene compounds have been isolated from the root bark of M. alba, few compounds are reported for their antimicrobial efficacy in vivo and the mechanism.The aim of the study was to isolate and identify compounds of the root bark of M. alba in view of their anti-MRSA bioactivity, evaluate the anti-MRSA bioactivity of compounds and 60% ethanol elution (MA-6) in vitro and in vivo, and explore preliminary antibacterial mechanism in order to provide natural resources against MRSA infection.Systematic phytochemical investigations were carried out according to the thin layer chromatography (TLC) of the active fraction MA-6 to find more anti-MRSA ingredients. The compounds of the root bark of M. alba were separated by column chromatography and identified by LC-MS/MS and NMR spectroscopy. The anti-MRSA efficacy of the active ingredients were evaluated by broth microdilution method and a murine infection model. The mode of action of compounds was explored by time-kill curve and post-contact effect. The preliminary mechanism of compounds against MRSA was explored by drug efflux pumps and bacterial biofilms.Chemical isolation resulted in twenty-nine known compounds, most with one or more geranyl and prenyl units exhibited superior anti-MRSA bioactivity, with MIC values of 2-16 μg/mL. In addition, the mode of action indicated that compounds presented persistent antimicrobial effect, which also produced concentration-dependent and time-dependent killing activity or property. Preliminary mechanism showed that the compound kuwanon O (29) damaged the bacterial cell membranes, leading to the accumulation of antibiotics inside bacterial cells, moreover, MA-6 and kuwanon O (29) inhibited the efflux of drugs by combining with methicillin or ethidium bromide (EtBr), resulting in the MICs of EtBr and methicillin were obviously decreased three-fold. The anti-MRSA efficacy in vivo indicated that the active fraction MA-6 could reduce bacteria in spleen, liver, kidney and mortality of acutely infectious mice, which was better than the positive drug berberine chloride.Experimental investigation showed that the MA-6 and compound 29 have promising bioactivity against MRSA in vitro and in vivo, which might be used as a potential source of new antibacterial medicine or a potential efflux pump inhibitor against MRSA infection.