## 3 Results and Discussion
### In Vitro Metabolite Identification in Rat and Human
\begin{table}
\begin{tabular}{c c c c c c c c c c c c} \hline \hline \multicolumn{1}{c}{**ID**} & \multicolumn{1}{c}{**Metabolic Reaction**} & \multicolumn{1}{c}{**Formula**} & \multicolumn{1}{c}{**RT**} & \multicolumn{1}{c}{**Exact**} & \multicolumn{1}{c}{**Mass**} & \multicolumn{1}{c}{**Error**} & \multicolumn{1}{c}{**Accurate**} & \multicolumn{1}{c}{**Fragment**} & \multicolumn{1}{c}{**Polarity**} & \multicolumn{1}{c}{**Rat**} & \multicolumn{1}{c}{**Human**} \\ \hline \multirow{4}{*}{Parent} & \multirow{2}{*}{Erinacine A} & & & & & & & & 119.0859, & & & & \\  & & & & & & & 199.1492, & & & & & \\  & & & & & & & 283.2055, & & & & & \\  & & & & & & & & 301.2162 \({}^{*}\) & & & & & \\  & & & & & & & & 69.0327, & & & & \\  & & & & & & & & 105.0709, & & & & \\ M1 & \multirow{2}{*}{Dehydration} & \multirow{2}{*}{C\({}_{25}\)H\({}_{34}\)O\({}_{5}\)} & \multirow{2}{*}{8.799} & \multirow{2}{*}{414.2399} & \multirow{2}{*}{\(-1.63\)} & \multirow{2}{*}{414.2406} & 119.0856 \({}^{*}\), & & & & & \\  & & & & & & & & 135.0817, & & & & \\  & & & & & & & & 235.1701, & & & & \\  & & & & & & & & 283.2036 & & & & \\  & & & & & & & & 97.0289, & & & & \\ M2 & \multirow{2}{*}{Demethylation} & \multirow{2}{*}{C\({}_{24}\)H\({}_{34}\)O\({}_{6}\)} & \multirow{2}{*}{4.916} & \multirow{2}{*}{418.2354} & \multirow{2}{*}{\(-0.25\)} & \multirow{2}{*}{418.2355} & 123.0551 \({}^{*}\), & & & & & \\  & & & & & & & & 231.0436, & & & & \\  & & & & & & & & 368.9803 & & & & \\  & & & & & & & & 55.0539, & & & \\ M3 & \multirow{2}{*}{Demethylation + Hydrogenation} & \multirow{2}{*}{C\({}_{24}\)H\({}_{36}\)O\({}_{6}\)} & \multirow{2}{*}{13.28} & \multirow{2}{*}{420.2488} & \multirow{2}{*}{\(-4.72\)} & \multirow{2}{*}{420.2512} & 77.0054 \({}^{*}\), & & & & & & \\  & & & & & & & 95.0854, & & & & \\  & & & & & & & & 147.1172, & & & \\  & & & & & & & & 277.2183, & & & \\  & & & & & & & & 353.2141 & & & \\  & & & & & & & 89.0597 \({}^{*}\), & & & & \\ M4 & 2 \(\times\) Hydroxylation & C\({}_{25}\)H\({}_{36}\)O\({}_{8}\) & 4.071 & 464.2405 & \(-1.41\) & 464.241 & 133.0860, & & & & & \\  & & & & & & & & 177.115, & & & & \\  & & & & & & & & 297.1842 & & & \\  & & & & & & & & 59.0601 \({}^{*}\), & & & \\ M5 & \multirow{2}{*}{Demethylation and two Hydroxylations} & \multirow{2}{*}{C\({}_{24}\)H\({}_{34}\)O\({}_{8}\)} & \multirow{2}{*}{8.777} & \multirow{2}{*}{450.2265} & \multirow{2}{*}{2.44} & 450.2254 & 101.0705, & & & & & & \\  & & & & & & & & 178.1225, & & & & \\  & & & & & & & 321.1516 & & & \\ \hline \hline \end{tabular}  
* Rat liver S9: M1 was detected at 10, 30 min, M2 and M3 were detected at 60 min, M4 and M5 were detected at 10, 30, 60 min. Human liver S9: M1, M2, M3, and M4 were detected at 10, 30, 60, 120 min, M5 was detected at 120 min.  
\end{table} 
Table 2: Common liver metabolites of erinacine A, detected in rat and human liver S9, with an UPLC-QTOF/MS. ions were _m/z_ 312.0488, 269.9469, 231.0432, 123.0552, and 179.0486 (Figure 4B). The product ion at _n/z_ 269.9469, 179.0486 corresponded to cleavage of the methylene (-CH3) and plus H+. The Demethylation + Hydrogenation metabolites (M3) formula was C24H36O6 with M2 reduction in alkene (+H2) [37; 38; 39]. The MS spectrum of M3 was found at [M + H]+ = 421.2569 and [M + Na]+ = 443.2355. At a collision energy of 40 V, the main product ions were _m/z_ 353.2141, 277.2183, 147.1172, and 77.0054 (Figure 4C). The product ion at _m/z_ 353.2141, 277.2183 were corresponding to Hydrogenation. The 2 \(\times\) Hydroxylation metabolites (M4) formula was C25H36O8 with erinacine A Hydroxylation of isopropyl side chain and cyclopentene [40; 41; 42]. The MS spectrum of M4 was found at [M + H]+ = 465.2461 and [M + Na]+ = 487.2315. At collision energy of 20V, the main product ions were _m/z_ 297.1842, 255.1389, 177.1115, 133.0860, and 89.0597 (Figure 4D). The product ion at _m/z_ 297.1842, and 89.0598 were corresponding to Hydroxylation. The Demethylation and two Hydroxylation metabolites (M5) formula was C24H34O8 with M11 losing a methylene (-CH2).