Study On The Active Constituents From The Root Bark Of Morus Alba L. Against Drug-resistance Bacteria

Objective:Our research group studied the chemical constituents from the root bark of Morus alba L.,especially the anti-MRSA（Methicillin-resistant Staphylococcus aureus）active compounds,and explored the mechanism of anti-MRSA of active compounds.Methods:（1）Extraction,separation and identification of chemical constituents:80%ethanol was used to extract constituents from the root bark of Morus alba L.Chromatographic separation technique was used to separate chemical components,such as MCI column chromatography,macroporous resin column chromatography,polyamide column chromatography,silica gel column chromatography,high performance liquid chromatography,and gel column chromatography.The compounds were purified,and the structures of the compounds were determined by¹H-NMR and ¹³C-NMR spectroscopy.（2）Anti-MRSA activity test and safety evaluation:A series of antibacterial activity tests on monomeric compounds were carried out,including K-B method,microbroth dilution method,time-kill curve,biofilm test and propensity to induce bacterial resistance.A preliminary safety evaluation was performed by hemolytic test.（3）Mechanism of action:The mechanism of anti-MRSA action was investigated by fluorescence microscopy,cytoplasma membrane depolarization,inner membrane permeabilization and RNA sequencing.Results and Conclusions:（1）A total of twenty monomeric compounds were obtained,eleven flavonoids:3’-geranyl-3-prenyl-2’,4’,5,7-tetrahydroxyflavone（1）,morusin（2）,cyclomorusin（3）,sanggenol A（4）,sanggenon N（5）,sanggenon I（6）,kuwanon U（7）,sanggenol P（8）,sanggenol L（9）,schizolaenone A（10）,sanggenol R（11）,and two Diels-Alder type adducts:mulberrofuran G（12）,albanol B（13）,and two terpenoids:lupeol（14）,ursolic acid（15）,and two steroids:β-sitosterol（16）,daucosterol（17）;and three other compounds:δ-tocopherol（18）,β-tocopherol（19）,14-hexadecenoic acid（20）.Among them,sanggenol R（11）is a new flavonoid,and schizolaenone A（10）was isolated from the root bark of Morus alba L.for the first time.（2）It was found that eleven flavonoids and two Diels-Alder type adducts had anti-MRSA activity by susceptibility test.Among them,the anti-MRSA activities of the compounds 3’-geranyl-3-prenyl-2’,4’,5,7-tetrahydroxyflavone（1）,sanggenon N（5）,sanggenon I（6）,kuwanon U（7）,sanggenol P（8）,sanggenol L（9）,schizolaenone A（10）,sanggenol R（11）were firstly discovered.Mulberrofuran G（12）,sanggenon N（5）,and sanggenon I（6）which were representative compounds were selected to be futher evaluated.Among them,mulberrofuran G had a continuous and effective bactericidal effect,sanggenon N and sanggenon I had a rapid bactericidal effect;mulberrofuran G,sanggenon N,sanggenon I can inhibit biofilm at a certain concentration and can effectively reduce the survival of bacteria in the biofilm;and they were difficult to induce bacterial resistance.Preliminary safety evaluation showed that mulberrofuran G,sanggenon N and sanggenon I had no significant effect on the stability of erythrocyte membrane at a concentration of less than or equal to16μg/mL.（3）The preliminary mechanism study of anti-MRSA showed:sanggenon N exerted bactericidal action through depolarization of cytoplasmic membrane;sanggenon I exerted bactericidal action by changing cell membrane permeability and plasmon membrane depolarization;mulberrofuran G exerted bactericidal action by acting on cell membrane-associated proteins.In summary,we can speculate that the main target of the active compounds tested is the bacterial cell membrane.