Influenza A (H1N1) Neuraminidase Inhibitors From Flos Caryophylli

Objective:A library of medicinal plant extracts was screened by high-throughput assay for in vitro inhibitory activity against influenza H1N1 neuraminidase(NA),and the MeOH extract of Flos Caryophylli was found to exhibit significant inhibitory effect.In the present study,we attempt to isolate and identify NA inhibitors from the MeOH extract of Flos Caryophylli.Methods:(1)Bioassay-guided fractionation was performed to isolate chemical components from Flos Caryophylli using various column chromatographies on silical gel and RP-HPLC;(2)Their chemical structures were then elucidated on the basis of spectroscopic data,including NMR and MS analysis and their physicochemical properties;(3)The cytopathic effect(CPE)reduction assay was carried out in MDCK cells to validate whether these isolates that exhibited anti-NA activities could protect host cells from influenza H1N1 virus infections.Results:(1)Nineteen compounds were isolated from the MeOH extract of Flos Caryophylli,and then identified as:rhamnetin-3-O-?-D-glucuronide-6"-methyl ester(ZN34023),rhamnazin-3-O-?-D-glucuronide-6"-methyl ester(ZN34024),rhamnazin-3-O-?-D-glucopyranoside(ZN34709),rhamnetin-3-?-D-glucopyranoside(ZN34708),isorhamnetin-3-O-?-D-glucopyranoside(ZN34705),isorhamnetin-3--?-D-glucuronide-6"-methyl ester(ZN34021),kaempferol-3-O-?-D-glucuronide-6"-methyl ester(ZN34020),quercetin 3-O-?-D-glucuronide-6"-methyl ester(ZN34018),quercetin 3-O-?-D-glucuronide(ZN32407),isoquercitrin(ZN34703),eugeniin(ZN40213),casuarictin(ZN40211),casuarinin(ZN40103),tellimagrandins I(ZN40104),1,3-di-O-galloyl-4,6-HHDP-glucose(ZN40106),1,2,3-tri-O-galloylglucose(ZN40203),1,2,3,6-tetra-O-galloylglucose(ZN40214),isobiflorin(ZN33905)and biflorin(ZN33204),respectively.Of them,two new compounds,rhamnetin-3-O-?-D-glucuronide-6"-methyl ester(ZN34023)and rhamnazin-3-O-?-D-glucuronide-6 "-methyl ester(ZN34024),along with three known compounds,rhamnetin-3-?-D-glucopyranoside(ZN34708),isorhamnetin-3-O-?-D-glucuronide-6"-methyl ester(ZN34021)and kaempferol-3-O-?-D-glucuronide-6"-methyl ester(ZN34020)have been isolated from this plant for the first time.(2)Among of the isolates,eugeniin showed the most potent inhibitory activity against NA with the IC50 values of 7.85 ?g/mL;Moreover,the most active compounds eugeniin was also found to protect MDCK cells from influenza infection with very low cytotoxicity to the host cells,its selective index(SI)in MDCK cells ranged from 81.65?254.42,which is higher than that of the reference Ribavirin.Conclusion:In summary,a series of neuraminidase inhibitors have been identified from the MeOH extract of Flos Caryophylli,and some of them were found to significantly inhibit the replication of influenza H1N1 viruses and exhibited low cytotoxicity to the host cells.The results have provided chemical templets of lead compounds for NA inhibitors of anti-influenza virus.