Rabbit conjunctivae edema and release of NO, TNF-α, and IL-1β from macrophages induced by fractions and esculentosides isolated from Phytolacca americana

Abstract

Context: The roots of Phytolacca americana L. (Phytolaccaceae) may be toxic. Despite heated controversy over the toxic compounds of P. americana, especially esculentosides, relevant studies remain scarce.Objective: The objective of this study is to screen the toxic fractions and compounds of P. americana, to determine the controlling indices, and to provide evidence for unraveling the mechanism.Materials and methods: Petroleum ether (PE), CH2Cl2, n-BuOH, and water fractions were isolated from 70% ethanol extract of P. americana. The n-BuOH fraction was dissolved in 50% ethanol and precipitated by adding ethyl ether. The resultant supernatants 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 esculentosides on rabbit conjunctivae (500 μg/eye) was observed by pathological examination and those on macrophages (5, 25, 50 and 100 μg/mL) were evaluated by detecting changes of NO, TNF-α, and IL-1β levels.Results and discussion: n-BuOH, SUP fractions, and EsC induced severe conjunctival edema. The four esculentosides induced dose-dependent releases of proinflammatory mediators NO, TNF-α, and IL-1β from macrophages, and releasing amounts peaked after 2 h of treatment. EsC and EsF induced macrophages to release mediators most significantly. EsC (50 μg/mL) functioned more effectively than EsF did, and similarly n-BuOH and SUPs fractions functioned more effectively than the esculentoside mixture. Thus, the four esculentosides exerted proinflammatory effects synergistically.Conclusion: All extracted esculentosides, especially EsC, induced inflammatory stimulation. Phytolacca americana-induced irritation of the gastrointestinal tract may be associated with esculentosides such as EsC.