## 1 Introduction  
The liver is considered the second-largest organ in the body [1]. After food and drugs enter the gastrointestinal tract through the oral cavity, the gastrointestinal pili will absorb digested nutrients and drugs. The portal vein system will receive the blood flow collected by the gastrointestinal tract and enter the liver for preliminary treatment of the nutrients, metabolites, and drug molecules [2, 3]. Metabolism is often divided into two phases of a biochemical reaction. Phase I involves oxidation, reduction, or hydrolysis by enzymes in the body. Phase II engages in the conjugation with small endogenous substances, turning them into highly soluble metabolites, enabling metabolites to be exported into the sinusoidal circulation for renal clearance, or into bile, which is then excreted from the body through urine or feces [4, 5, 6]. Metabolite identification in the early stage of drug discovery is necessary to assess the knowledge for improving pharmaceutical property, safety, and efficacy of the bioactive molecule. Identification of metabolites and reactive intermediates suggest the need for structural modifications in investigational compounds to avoid subsequent toxic consequences. Even if the drug has been on the market, identification of the metabolites and evaluation is required to avoid the risk of their post-marketing withdrawal [7,8]. Therefore, in vitro, S9 fractions analysis provides several advantages compared to time-consuming in vivo studies: (1) they are amenable to high throughput screening and automation; (2) using S9 fraction allows for the testing of large quantities of compounds for drug discovery in a short period; (3) helps to reduced animal usage [9].  
_Hericium erinaceus_ (HE) is an edible mushroom that inhabits mountainous areas of the northeast territories in Asia [10], Europe, and North America [11]. HE belongs to Basidiomycota (Phylum), Agaromycetes (Class), Russuales (Order), Hericiaceae (Family), and Hericium (Genus). HE has been documented to display a wide range of beneficial properties, including anti-oxidant, anti-tumor, anti-aging, hypolipidemic, and gastric mucosal protection effects [12, 13, 14]. In 1994, [PERSON] et al. discovered that the diterpenoid erinacine A, B, and C, in the mycelium of HE, could promote the production of stellate cell lines in the rat brain and the stimulation of nerve growth factor (NGF) synthesis [15]. Erinacine A can confer neuroprotective effects and attenuates oxidative stress against stroke [16], Alzheimer's disease [17], Parkinson's disease [18], ischemic stroke, and depression in vivo [19,20]. Moreover, erinacine A can pass through the blood-brain barrier of rats to support the development of HE mycelia as a functional food for neuro health improvement [21]. This edible mushroom mycelium has become a hot issue with researchers attempting to understand its important role in the central and peripheral nerves' development, differentiation, growth, and regeneration [22]. Recent studies have also showed that erinacine A enriched HE has potential benefits in treatment of Alzheimer's and Parkinson's disease [23, 24, 25]. However, no study has discussed erinacine A's possible metabolic biomarkers that may contribute to this nutraceutical effects.  
In this study, erinacine A compound was purified from the erinacine A enriched HE mycelium and was metabolized using two different liver S9 fractions: rat and human. Using liquid chromatography/triple quadrupole mass spectrometer (HPLC-QQQ/MS) for quantitative analysis of erinacine A, and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) to identify the metabolites of erinacine A at each time point. We aim to understand the metabolic rate of erinacine A and establish its metabolite profile database to help promote the application of nutraceutical supplements and the research of potential medication in the future.