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The objective of this study was to evaluate whether silk fibroin (SF) incorporated into 4-hexylresorcinol (4HR) could increase botulinum toxin-A (BTX-A) activity.In total, 30 rats were used for this study. The animals were divided into 6 groups according to the injected materials (SA: saline only; SF; 4HR; B2: 2 units of BTX-A; B2 + SF + 4HR: combination of B2, SF, and 4HR; B5: 5 units of BTX-A). Serial sonography was used for the evaluation of muscle thickness after injection. Immunohistochemical staining was used for the evaluation of myosin type II (myo2) and Bcl-2 protein expression.The relative thickness of the masseter muscle in B2 group was 66.14% ± 4.55% to the preinjection level; in B2 + SF + 4HR group was 54.59% ± 4.83%, and in B5 group was 56.19% ± 8.28%. Any BTX-injected group showed significantly lower value of the relative muscle thickness compared to SA, SF, or 4HR group (P < 0.001 for all). The difference of relative muscle thickness between B2 group and B2 + SF + 4HR group was statistically significant (P < 0.001). The intensity of myo2 immunostaining in B5, B2, and B2 + SF + 4HR group was significantly higher than those in the other groups (P < 0.05).When 2 units of BTX was incorporated to SF and 4HR, combination formula showed similar activity to those of 5 units of BTX.