Investigating the scientific basis behind the ancient processing method of heshouwu by hepatotoxicity assessment and chemical characterization

Processing is a unique feature in the application of Chinese medicinal materials (CMMs), and major objectives of the processing are to reduce toxicity and/or enhance efficacy. However, as technology has changed and as demand has increased, processing methods have changed greatly. It is not clear whether the change of processing methods alters the chemistry and thus toxicity and efficacy of CMMs. Heshouwu, the tuberous root of Polygonum multiflorum Thunb., has been widely reported as having hepatotoxicity recently. According to the Ben Cao Gang Mu, heshouwu should be steamed and sun-dried for nine cycles after peeling off the coarse cork and cortex. Nevertheless, the current processing method of heshouwu as documented in the Chinese Pharmacopeia is stewing or steaming for only one cycle and without peeling off the cork and cortex. Our recent study has demonstrated that there are significant differences in the chemical profiles of heshouwu processed using the modern method and the ancient method. These facts lead to several questions: Could the hepatotoxicity reported for heshouwu today derive from modern processing method used? Are the cork and cortex of heshouwu the major tissue parts responsible for hepatotoxicity? Is the ancient processing method and how to detoxify heshouwu?

In this study, we will address these questions. First, in order to find out the principle of peeling off the cork and cortex, laser microdissection (LMD), UHPLC-QTOF-MS/MS and UPLC-QqQ- MS/MS will be used to map the distribution patterns of secondary metabolites in different plant tissues of raw heshouwu; the hepatotoxicity of each tissue part of raw heshouwu will be evaluated in vitro. Second, in order to further explore the principle, the coarse bark (i.e. cork and cortex) and the rest part of raw heshouwu will be used for hepatotoxicity screening in vitro and in vivo as well as the chemical comparison. Finally, in order to reveal whether the modern one- cycle processing method is responsible for heshouwu’s hepatotoxicity, and whether and how the traditional nine-cycle processing method reduces hepatotoxicity of heshouwu, hepatotoxic changes and chemical variation between raw and processed heshouwu from each processing cycle of the ancient method and modern method will be assessed, thereby the correlation between processing-induced hepatotoxic changes and processing-induced variation of secondary metabolites in heshouwu will be revealed. This work will provide scientific basis behind the ancient processing method of heshouwu, thereby offering evidence to standardize the processing method of heshouwu to ensure its safety.