| 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 EtOAc extract of Polygonum cuspidatum was found to exhibit significant inhibitory effect. In the present study, we attempt to isolate and identify small-molecule NA inhibitors from the extract of P. cuspidatum.Methods:① We first evaluated the ability of 236 extracts (90 species of medicinal plants, belong to 48 families) to inhibit NA activity by high-throughput screening assay;② Bioassay-guided fractionation was performed to isolate chemical components from P. cuspidatum by using various column chromatographies on silical gel, Sephadex LH-20 and HPLC;③ Their chemical structures were then elucidated on the basis of spectroscopic data, including NMR, MS, IR and UV analysis and their physicochemical properties;④ 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; ⑤ The structural modifications of NA inhibitor resveratrol were performed to study structure-activity relationships.⑥ Micro-fraction KT53701, which contains the NA inhibitors, was prepared by preparative RP-HPLC. And we further examined inhibitory effects of micro-fraction KT53701 on influenza H1N1 virus.Results:① 236 extracts from 90 medicinal plants were screened for in vitro inhibitory activity against neuraminidase. Among the tested extracts,14 extracts significantly inhibit NA activity, the EtOAc extract of P. cuspidatum was found to exhibit significant inhibitory effect with the value of IC50= 29.8 μg/mL; ② Twelve compounds were isolated from the EtOAc extract of P. cuspidatum and identified as: 6-Acetyl-5-hydroxy-2-ethoxy-7-methyl-1,4-naphthoquinone (KT53504), emodin (KT34501),2-methoxystypandrone (KT45004), resveratrol (KT50105), polydatin (KT44802), emodin-8-O-β-D-glucopyranoside (KT44905), polydatin-2’-O-gallate (KT50208), catechin-3-O-gallate (KT51711), torachrysone-8-O-β-D-glucopyranoside (KT53105), citreorosein (KT54108) and 4,6-dihydroxybenzofuran-3-one (KT50503), respectively. Of them, compound KT53504 is a new naphthoquinone; ③ Among of the isolates, catechin-3-O-gallate, together with polydatin-2’-O-gallate and resveratrol, showed potent inhibitory activity against NA with the IC50 values of 9.4,24.3 and 29.6 μg/mL, respectively. Moreover, the most active compounds catechin-3-O-gallate and polydatin-2’-O-gallate were found to protect MDCK cells from influenza infection (ECso= 0.4 and 3.2 ug/mL) with very low cytotoxicity to the host cells, their selective index (SI) in MDCK cells ranged from 289 and 57, which is higher than that of the reference Zanamivir; ④ Seven derivatives have been prepared by structural modifications of NA inhibitor resveratrol, further studies are underway to evaluate their anti-virus activities;⑤ HPLC fingerprinting of micro-fraction KT53701 has shown the presence of NA inhibitors such as catechin-3-O-gallate, resveratrol and polydatin-2’-O-gallate. Micro-fraction KT53701 significantly inhibited the NA activity and the replication of influenza HlNl viruses, and exhibited low cytotoxicity to the host cells.Conclusion:In summary, we examined medicinal plant extracts for in vitro NA inhibitory activity by high-throughput screening assay, and a series of neuraminidase inhibitors have been identified from the EtOAc extract of P. cusp/datum. And some of them were found to significantly inhibit the replication of influenza H1N1 viruses and exhibit low cytotoxicity to the host cells. The results have provided the chemical basis for understanding of effective components responsible for anti-influenza virus activities in this plant. |
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