## 4 Discussion  
Issues related to sensitive skin are increasing due to environmental and lifestyle changes. The associated symptoms, such as itching, stinging, burning, and pain, can severely reduce the quality of life. Therefore, it is important that effective cosmeceuticals are developed to ameliorate the symptoms of sensitive skin. In the present study, TRPV1--a receptor that is activated in sensitive skin--was used as a target to discover natural bioactive compounds that could reduce irritation by capsaicin--a TRPV1 agonist. TRPV1 is a cation channel that promotes calcium influx when activated; thus, we confirmed the activation and inhibition of TRPV1 through calcium imaging. Increased calcium levels following treatment with 10 uM capsaicin proved that TRPV1 was activated without abnormality in the cell model. Troxerutin significantly blocked capsaicin-induced TRPV1 activation, suggesting that troxerutin could be a potential antagonist that inhibits the effects of stimuli, such as chemical contact, itching, and pain, on skin sensitivity. Troxerutin is a flavonoid found in a variety of plants, including the Japanese pagoda tree _Sophora japonica_. Troxerutin exhibits various beneficial activities, such as free radical scavenging [32], anti-arrhythmogenic and anti-inflammatory effects against ischemia/reperfusion injury of diabetic myocardium [33], blood pressure and lipid metabolism regulation [34], and elastase inhibition resulting in anti-inflammatory effects [35]. Although previous reports support the phenotypic efficacy of troxerutin against certain diseases, the target protein-based pharmacological interaction remains unclear due to its multimodal effects [36]. To our knowledge, the present study is the first to identify the antagonistic effects of troxerutin against TRPV1 using ML, which could promote target-based mode-of-action studies of troxerutin. Troxerutin has been widely studied in drug development owing to its oral bioavailability and low toxicity (rat LD50 = 27 160 mg/kg). However, previous clinical studies reported a maximum plasma concentration (Cmax) of 2931 \(\pm\) 1018 pg/mL following oral administration of 300 mg troxerutin, which makes it challenging to develop troxerutin as an orally administered drug or supplement against a wide range of indications [14,37]. Although the oral administration route may be insufficient, a study using a stripping method with troxerutin showed an excellent skin permeation rate of 80%, suggesting that troxerutin possesses sufficient competitiveness as a cosmeceutical compound, consistent with our hypothesis [32]. The interaction between troxerutin and TRPV1 seems to bear an effective concentration between 50 ug/mL and 100 ug/mL (67-134 uM) in the presence of 10 uM capsaicin. Based on the \(K_{D}\) and IC50 values estimated in the DTI model, future studies could potentially measure \(K_{D}\) and IC50 values experimentally via surface plasmon resonance, isothermal  
Figure 5: Immediate soothing effect of troxerutin after stimulation with 0.1% \(w/v\) capsaicin. (**a**) Variation in skin redness immediately after treatment with troxerutin lotion. Data are presented as the mean \(\pm\) standard deviation (SD). NS, not significant; * \(p\) < 0.05 compared to the vehicle group at T3. (**b**) Soothing rate following application of lotions. Data are presented as the mean \(\pm\) standard error (SE). * \(p\) < 0.05 compared with the vehicle group. titration calorimetry, microscale thermophoresis, or patch-clamp assays to accurately examine the relationship between predictions and wet-experiment results. The TRPV1 response inhibition rate of troxerutin was \(\sim\)80% at 134 uM; however, the predicted values of the DTI model were not directly correlated to the experimental values. The accuracy of the ML model could potentially be improved by additional drug-target data feed-in. In the structural modeling experiment, troxerutin and JTS-653 were found to have similar docking positions at the binding pocket of hTRPV1.