## 3 Results
### Effect of Active Ingredient on Cantileies  
The second step of our investigation was to identify whether our active ingredient was able to act globally on all pathways impacted by the process of canities. We evaluated the gene expression after incubation for 48 h with the active ingredient at 1% (\(\
u\)/\(\
u\)) on scalp skin explants under oxidative stress exposure mimicking the canities model. In Figure 4, the results demonstrated that after the treatment with the active ingredient, the genes encoding for melanogenesis were up-regulated (EDN1, MC1R, MITF, POMC). The formation of melanosomes and their transfer were reactivated, as a result of the up-regulation of AP3B1, CTNS, HPS5, KRT5 and MYO5A expressions. The expressions of genes involved in renewal (FST, KRT19) and autophagy (MAP1 LC3A) were also up-regulated. Finally, the treatment with the active ingredient showed a significant stimulation of genes involved in antioxidant activities (HMOX1, GLRX, GSS, MGST1, NRF2 and TXN). These results suggested oxidation level control and boosting of the melanin synthesis pathway. To further our work, we used the microdissected hair follicle canities-induced model to evaluate the effect of the active ingredient on redox balance control and melanocyte reservoir protection using ROS quantification and NKI/beteb immunostaining. In Figure 5, we observed that, in the presence of the active ingredient at 1%, the ROS production was significantly reduced by 54%. In Figure 6, the presence of the active ingredient demonstrated a significant protection of melanocyte reservoirs against oxidative stress, as observed with the increase in the NKI/beteb-positive cell number by 190%. These results confirmed that the active ingredient was able to neutralise the high level of oxidative stress and thus protected the melanocyte reservoirs in hair follicles. Figure 4: Gene expression analysis in scalp skin explants in a canities-induced condition following treatment with the active ingredient at 1%. The results (mean \(\pm\) SEM) are expressed as relative expression versus the oxidative stress control condition. Statistical analysis was performed using one-way ANOVA followed by [PERSON]’s \(t\)-test in comparison with oxidative stress control condition, with # \(p\) < 0.1, * \(p\) < 0.05, ** \(p\) < 0.01 and *** \(p\) < 0.001. Figure 5: ROS production assessment in hair follicles in a canities-induced condition (cumene hydroperoxide at 50 \(\upmu\)m) in the presence of active ingredient at 1%. (**A**) Representative images. Magnification: 20\(\times\). (**B**) Quantification of the mean fluorescence intensity (\(\pm\) SEM) of DCFH-DA probe. Statistical analysis was performed using one-way ANOVA with permutation test followed by [PERSON]’s test, with **_p_ < 0.01. The final expectation of the active ingredient was for it to reactivate the pigmentation in a natural canities condition. The melanin content of grey hair follicles was evaluated by  
Figure 6: Melanocyte and melanoblast quantification by NKI/beteb immunostaining in a canities-induced condition (cumene hydroperoxide at 50 μm) in the presence of active ingredient at 1% in hair follicles. (**A**) Representative images. Magnification: 10\(\times\). (**B**) Data are presented as mean \(\pm\) SEM. Statistical analysis was performed using one-way ANOVA with permutation test followed by a \(t\)-test with permutation, with **_p_ < 0.01. Fontana-Masson staining. In Figure 7, the results of melanin content quantification showed a significant increase in production of melanin by 15% after 3 days of treatment in presence of the active ingredient at 1%. Thanks to these models developed to mimic canities, we demonstrated that our active ingredient was able to protect hair follicles against oxidative stress, preserve stem cells and melanocytes and reactivate melanogenesis. These first steps led to a total control of the main causes of canities development and promoted restoration of hair follicle pigmentation. We then carried out a clinical study with double-blind and placebo-controlled conditions.