Preliminary Research On The Metabolomics Of Toxic Saponins And The Process Of Reducing Toxicity In Phytolacca Radix

Phytolacca radix is a common traditional Chinese medicine,derived from the dried roots of Phytolacca acinosa Roxb and Phytolacca americana L.It is included in the Chinese Pharmacopoeia（2020 edition）for its important medicinal value.Due to its side effects,it has been recorded as toxic since ancient times.Many poisoning incidents occur due to the similarity of Phytolacca radix to other edible or medicinal plant roots,which can lead to accidental ingestion.The toxicity of Phytolacca radix is a limiting factor for its widespread clinical use.However,there is no direct and definitive evidence to show which ingredient is the toxic culprit.In this study,the main toxic components of Phytolacca radix were firstly obtained by bioactivity-directed separation through various chromatographic techniques（column chromatography,high performance liquid chromatography,etc.）,using the mortality of zebrafish larvae as the index of toxicity investigation.The structure of the compound was identified as Esculentoside B by ¹H NMR,¹³C NMR,and HR-MS.Subsequently,the toxicity of Esculentoside B to zebrafish larvae was further assessed using zebrafish as a toxicological model in this study.This study found that Esculentoside B exposure at sublethal concentrations(LC₁₀)causes abnormal development and neurotoxicity in zebrafish larvae.Meanwhile,this study used LC-MS/MS metabolomics analysis to explore the effects of Esculentoside B exposure on metabolites in zebrafish larvae.We found that Esculentoside B exposure led to the disruption of metabolites in zebrafish.23 differential metabolites were screened,of which 11 differential metabolites could be enriched to pathways,and four metabolic pathways（D-amino acid metabolism,protein export,autophagy,and m TOR signaling pathways）were altered.These metabolic pathways directly or indirectly affected the function of the nervous system.In addition,this study also investigated the effect of fermentation treatment of eight edible bacteria on the content of the main toxic components of Phytolacca radix.The results showed that Saccharomyces cerevisiae BV818 significantly increased Esculentoside B content,while Acetobacter pasteurianus HU1.01 significantly decreased Esculentoside B content.The result suggested that Phytolacca radix could be fermented using Acetobacter pasteurianus to obtain a less toxic version of Phytolacca radix.This study was the first to confirm that Esculentoside B was the most important toxic constituent of Phytolacca radix.Also,Esculentoside B was found to be neurotoxic for the first time in this study.Our results provide a theoretical basis for further studies on the neurotoxic effects and potential molecular mechanisms of Esculentoside B and lay the foundation for further development and consumption of Phytolacca radix.