Isolation, Structure Identification And Pharmacological Studies Of STAT3Signaling Pathway Inhibitors From Zanthoxylum Nitidum (Roxb.) DC.

Objective:The aim of the present study was to identify novel small-molecule inhibitors of STAT3signaling pathway from the extracts of the roots of Zanthoxylum nitidum (Roxb.) DC. and further examine in vitro and in vivo anti-cancer and anti-inflammatory activities of small-molecule inhibitors of STAT3signaling pathway. Methods:①The different extracts and subftractions of Z. nitidum were screened by using a STAT3-reporter gene assay;②Bioassay-guided fractionation was performed to purify chemical components from Z.nitidum by using various column chromatographies on silical gel, Sephadex LH-20, PTLC and HPLC;③Their chemical structures were then elucidated on the basis of spectroscopic data, including NMR and MS analysis and their physicochemical properties;④We further examined the inhibitory potencies and the specificity of the isolates on multiple signaling pathways;⑤We moreover examined whether small-molecule STAT3inhibitors are effectively in inhibiting cell proliferation, cell survival, migration and invasion of human cancer (stem) cells, and evaluate in vivo anti-cancer efficacy of STAT3inhibitors in human cancer xenograft models;⑥Experimental burn model was carried out to validate whether the small-molecule STAT3inhibitors exhibit anti-inflammatory activities and promote burn wound healing in mice. Results:①The EtOAc and MeOH extracts of Z. nitidum was found to exhibit significant inhibitory effect on IL6/STAT3signaling pathway;②Twelve compounds were isolated from the extracts of Z. nitidum, and eleven of the isolates were identified as:8-methoxy-dihydrochelerythrine (YS12806),8-methoxy-9-demethoxyldihydrochlerythrine (MH24701), oxychelerythrine (MH23711), avicine (MH24506), nitidine (MH24218), liriodenine (MH24404), isobutyl benzoate (MH32501), L-sesamin (YS13805), skimmianine (YS13206), β-stigmasterol (YS13806) and4-hydroxy-N-methylproline (YS12903), respectively;③8-methoxy-dihydrochelerythrine (YS12806) demonstrated a potent and selective inhibitor of IL-6/STAT3signaling pathway (IC50=7.1μM), inhibited cell proliferation, cell migration and invasion, and induced cell apoptosis of human cancer (stem) cells (IC50=0.22-8.0μM); YS12806was also found to significantly attenuate JAK2and STAT3phosphorylation (p-JAK2and p-STAT3) and markedly reduced the protein level of STAT3downstream targets Cyclin B1、Survivn、ICAM-land VEGF in Huh-7cells;④Treatment with YS12806at20mg/Kg dramatically decreased the tumor volumes of Huh-7cell line-derived xenografts in mice;⑥YS12806significantly promoted burn wound healing in mice. Conclusion:In the present study, eleven compounds were identified from the extracts of the roots of Z. nitidum by using a STAT3-reporter gene assay, and compounds avicine, isobutyl benzoate and4-hydroxy-N-methylproline are obtained from this plant for the first time;8-Methoxy-dihydrochelerythrine (YS12806) demonstrated a potent and selective inhibitor of IL-6/STAT3signaling pathway and has provided chemical template as molecular targeted anti-cancer agents.