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Context: The roots of Phytolacca americana L.(Phytolaccaceae) may be toxic due to considerable side effects.Despite heated controversy over the toxic compounds of P.americana, especially esculentosides, relevant studies remain scarce.Objective: To screen the toxic fractions and compounds of P.americana and to determine the controlling indices,and to provide evidence for unraveling the mechanism.Materials and methods: Petroleum ether (PE), CH2C12, n-BuOH and water fractions were isolated from 70% ethanol extract of P.americana.The the n-BuOH fraction was dissolved in 50% ethanol and precipitated by adding ethyl ether.The resultant supernatant and precipitates were referred to as SUPs and SEDs fractions respectively.SUPs fraction was separated by column chromatography into four main stimulating esculentosides that were identified by HR-ESI/MS and NMR as EsA,EsB, EsC and EsF.The irritating effects of all fractions and esculentosides on rabbit conjunctivae and macrophages were evaluated.Results and discussion: n-BuOH and SUPs fractions induced rabbit conjunctival edema and NO release from macrophages more evidently than other fractions did.EsC induced rabbit conjunctival edema and release of proinflammatory mediators NO, TNF-α and IL-lβ from macrophages most effectively, which was followed by EsF.In other words, EsC had higher toxicity than EsA, EsB, and EsF did.Since the mixture of the four compounds exhibited similar toxicity to EsC did, they exerted proinflammatory effects synergistically.Conclusion: All extracted esculentosides, especially EsC, induced inflammatory stimulation.Macrophage-secreted TNF-α and NO can induce mucosal barrier dysfunction and diarrhea.Therefore, P.americana-induced irritation of the gastrointestinal tract may be associated with esculentosides such as EsC.