## 3 Results and Discussion
### In Vitro Metabolite Identification in Rat and Human
The MS spectrum of M5 was found at [M + H]+ = 451.2413 and [M + Na]+ = 473.2158. At collision energy of 40 V, the main product ions were _m/z_ 321.1516, 178.1225, 101.0705, and 59.0601 (Figure 4E). The product ion at _m/z_ 321.1516 corresponded to M4 cleavage of the methylene (-CH3) and plus H+. There have been 70 different secondary metabolites isolated from either HE sporophore or mycelium. These metabolites are identified as polysaccharides, polyketides, phenols, terpenoids, and bioactive molecules [43; 44; 45]. We speculated that these common metabolites, which we analyzed, might also be the precursors or intermediate products of the mycelium secondary metabolites mentioned before. Nevertheless, it has been reported that M1, M2, M3, M4, and M5 may represent key intermediates between erinacine A and secondary metabolites for rat and human studies. Due to the increasing use of HE mycelium as a functional supplement, identifying these metabolites will also reveal useful biomarkers. Taking cholesterol as an example, the precursors or intermediate products of various substances, produced by the liver enzymes process, were metabolized to oxygentol, which is now understood as a potent metabolite in sterol metabolism and synthesis regulation [46]. Figure 2: Proposed metabolic pathway of erinacine A.  
Figure 4: _Cont._  
Figure 3: **(A) MS spectrum of erinacine A with an ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry. (B) MS/MS spectrum of erinacine A with an ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry. (C) The proposed structures of product ions, produced by erinacine A, at a collision energy of 10 V. (a) [C\({}_{20}\)H\({}_{29}\)O\({}_{2}\)]\({}^{+}\) = 301.2162, (b) [C\({}_{20}\)H\({}_{27}\)O]\({}^{+}\) = 283.2052. \(\blacklozenge\):** Parent ion. Figure 4: _Cont._  
M2 and M3 were detected at 60 min for rat liver S9, whereas for human liver S9, M2 and M3 were detected at 10, 30, 60, and 120 min. We conjectured that M3 might be produced by two distinct metabolic pathways; one from erinacine A to M3, and the other was derived from M1. In contrast, M4 and M5 were detected at 10, 30, and 60 min rat liver S9. In human liver S9, M4 was detected at 10, 30, 60, and 120 min. M5 was detected at 120 min. The time of appearance can explain that M5 metabolic pathway may be derived from M4. This study provides additional information for the HE mycelium clinical studies missing information, which is the metabolic action from the bioactive compound. In a recent randomized, double-blind placebo-controlled study, three 350 mg/g HE mycel (containing 5 mg/g erinacine A) capsules intervention, for 49 weeks, demonstrated higher Cognitive Abilities Screening Instrument, Mini-Mental State Examination, and Instrumental Activities of Daily Living scores and achieved a better contrast sensitivity, in patients with age > 50 years old and diagnosis of probable Alzheimer's Disease, when compared to the placebo group [19]. By understanding erinacine A metabolites profile, we can interpret that erinacine A enriched HE mycelia improved neurocognitive disorder, which might be contributed mostly from the unmetabolized compound. As for erinacine A, metabolites are well-tolerated and might be important in achieving neuro-nutritional benefits, which will be investigated in future studies.