## 3 Results
### Inhibition of Fatty Acid Anandamide Hydrolase  
In order to check whether the HPE extracts were able to affect the fatty acid amide hydrolase (FAAH), we analyzed both the FAAH mRNA expression level and enzymatic activity. HPE\({}_{\text{H2O}}\) was able to decrease the FAAH mRNA level even if the downregulation was not statistically significant, whereas all other extracts were ineffective (Figure 5A). Interestingly, under our experimental conditions, all the extracts were able to interfere with the FAAH activity, although with different efficacy and potency (Figure 5B). Particularly, HPE\({}_{\text{H2O}}\) was the least effective sample, achieving a maximum 58.5% enzyme inhibition at the highest concentration of 2500 \(\upmu\)g/mL. Conversely, the other extracts could completely inhibit the FAAH enzyme, HPE\({}_{\text{EOH100}}\) being slightly more potent than HPE\({}_{\text{DMSO}}\) and HPE\({}_{\text{EOH50}}\), which displayed similar potencies. Indeed, the IC\({}_{\text{50}}\) value of HPE\({}_{\text{EOH100}}\) was about 1.2- to 1.5-fold lower than those of HPE\({}_{\text{EOH50}}\) and HPE\({}_{\text{DMSO}}\) (Table 4). Under the same experimental conditions, 100 \(\upmu\)g/mL harpagsoside (corresponding to 200 \(\upmu\)M) was found to be ineffective in the inhibition of the FAAH enzyme (about 9% inhibition compared the control). Conversely, the positive control JZL 195 (20 \(\upmu\)M corresponding to 8.7 \(\upmu\)g/mL) produced a maximum 90% enzyme inhibition (Table 4). As expected, the positive control was significantly more potent than the HPE extracts (Table 4).  
\begin{table}
\begin{tabular}{c c} \hline \hline _Harpagophytum procumbens_ **Root Extract** & **IC\({}_{\text{50}}\) (CL) \(\upmu\)g/mL** \\ \hline HPE\({}_{\text{EOH30}}\) & 94.7 (23.8–97.5) \\ HPE\({}_{\text{EOH100}}\) & 65.5 (19.2–87.9) * \\ HPE\({}_{\text{EOH50}}\) & 73.8 (24.5–94.3) \\ HPE\({}_{\text{EHO}}\) & - \\ JZL 195 & 0.03 (0.01–0.06) * \\ \hline \hline \end{tabular}  
* Not evaluable being lower than 80% inhibition achieved.
* \(p<0.05\) significantly lower than HPE\({}_{\text{DMSO}}\) (ANOVA followed by [PERSON]’s multiple comparison post hoc test).  
\end{table}
Table 4: IC\({}_{\text{50}}\) values of _Harpagophytum procumbens_ root extract (HPE) dissolved in DMSO (HPE\({}_{\text{DMSO}}\)), 100% \(\upnu\)g/\(\upnu\) EtOH (HPE\({}_{\text{EOH100}}\)), 50% \(\upnu\)g/\(\upnu\) EtOH (HPE\({}_{\text{EOH50}}\)), and deionized water (HPE\({}_{\text{H2O}}\)) and the positive control JZL 195 in the FAAH inhibition assay.  
Figure 5: Effects of _Harpagophytum procumbens_ root extract (HPE) on fatty acid anandamide hydrolase (FAAH) expression and enzymatic activity. (**A**) Cells were treated with 0.1 mg/mL of _Harpagophytum procumbens_ root extract (HPE) dissolved in deionized water (HPE\({}_{\text{H2O}}\)), DMSO (HPE\({}_{\text{DMSO}}\)), 100% \(\upnu\)g EtOH (HPE\({}_{\text{EOH100}}\)), and 50% \(\upnu\)g EtOH (HPE\({}_{\text{EOH50}}\)), for 24 h. Cells were then harvested, and mRNA was extracted and analyzed by RT-PCR. FAAH mRNA levels were reported as relative mRNA expression level with respect to 18S mRNA (2–\(\Delta\)ACt method). Results are expressed as mean \(\pm\) S.E.M. of data obtained by three different experiments. (**B**) Concentration–response curves showing the inhibitory effects on FAAH from HPE\({}_{\text{DMSO}}\), 100% \(\upgamma\)/\(\upnu\) HPE\({}_{\text{EOH100}}\), 50% \(\upgamma\)/\(\upnu\) HPE\({}_{\text{EOH50}}\), and HPE\({}_{\text{H2O}}\). Data are the mean \(\pm\) SE of at least three independent experiments with two replicates for each experiment (n = 6).