One-sixth of the currently known natural products contain α,β-unsaturated carbonyl groups.Our previous studies re-ported a rare C-sulfonate metabolic pathway.Sulfonate groups were linked to the β-carbon of α,β-unsaturated carbonyl-based natural compounds through this pathway.However,the mechanism of this type of metabolism is still not fully understood,especially whether it is formed through enzyme-mediated biotransformation or direct sulfite addition.In this work,the enzyme-mediated and non-en-zymatic pathways were studied.First,the sulfite content in rat intestine was determined by LC-MS/MS.The results showed that the amount of sulfite in rat intestinal contents was from 41.5 to 383 μg·g-1,whereas the amount of sulfite in rat feed was lower than the lower limit of quantitation(20 μg·g-1).Second,the reaction kinetics of sulfite-andrographolide reactions in phosphate buffer solutions(pH 6-8)was studied.The half-lives of andrographolide ranged from minutes to hours.This was suggested that the C-sulfonate reac-tion of andrographolide was very fast.Third,the C-sulfonate metabolites of andrographolide were both detected when andrograph-olide and L-cysteine-S-conjugate andrographolide were incubated with the rat small intestine contents or sulfite,indicating that the sulfite amount in rat intestine contents was high enough to react with andrographolide,which assisted a significant portion of andro-grapholide metabolism.Finally,the comparison of andrographolide metabolite profiles among liver homogenate(with NADPH),liver S9(with NADPH),small intestine contents homogenate(with no NADPH),and sulfite solution incubations showed that the C-sulfon-ate metabolites were predominantly generated in the intestinal tract by non-enzymatic pathway.In summary,sulfite can serve as a sub-strate for C-sulfonate metabolism,and these results identified non-enzymatically nucleophilic addition as the potential mechanism for C-sulfonate metabolism of compounds containing α,β-unsaturated carbonyl moiety.