Effects of the healing activity of rosemary-of-Chapada (Lippia gracilis Schauer) on cutaneous lesions in rats

Abstract

Purpose: To evaluate the effects of rosemary leaf essential oil-based ointments on the healing of rat skin lesions.

Methods: Sixty adult male rats, with dorsal excisional skin wounds made surgically under anesthesia, were divided into three groups (n = 20): Sham group (untreated wounds); control group (CG, wounds treated with vehicle); and essential oil (EO) treated group (wounds treated with essential oil-based ointments), administered topically once daily. Skin wounds were evaluated at 4, 7, 14, and 21 days after EO or vehicle treatments. Lesions were analyzed macroscopically for the contraction degree. Formalin-fixed paraffin-embedded sections of skin wounds were used for histopathological evaluation.

Results: Macroscopic evaluation showed wounds edges with thin crust without firmness and yellowish color, along with an improvement in wound contraction in EO group when compared to the other groups. A reduced inflammatory reaction, along with newly formed small diameter capillaries and more organized and elongated collagen fibers, were more frequently observed in EO group than in the other groups. Moreover, blood vessel number and collagen fibers density were significantly higher in EO group.

Conclusions: Skin lesion treatment with rosemary leaf essential oil-based ointments accelerates the initial stages of healing, reduces inflammation, and increases angiogenesis, collagen fibers density, and wound contraction in rats.

Figure 1. (a) Chemical composition of the essential oil obtained from L. gracilis Schauer leaves. Compounds are listed in order of election in a nonpolar DB-5 column; retention indices calculated through the retention times in relation to the n-alkanes series (C9-C19) in a Phenomenex ZB5-MS 30 m × 0.25 μ column; % Relative: relative percentage of the compound. (b) GC-MS chromatogram of the essential oil obtained from L. gracilis Schauer leaves.

Figure 2. Wound contraction was improved in the group treated with essential oil from L. gracilis leaves (EO). (a) Wound closure kinetics in EO, CG and Sham groups. (b) Representative macroscopic images of the wounds from each group. Wound contraction results were expressed as percentage of closure in relation to the original size (1 – [wound area] / [original wound area] × 100). Data are represented as mean ± SEM; n = 5 rats in each time point. *** p < 0.001, on the 7th postoperative day (Two-way ANOVA).

Figure 3. Angiogenesis was increased in the essential oil treated group (EO). (a) Kinetics of blood vessel count. (b) Representative photomicrographs of H/E-stained histological sections 7 days after wounding. Arrows indicate blood vessels. Data are expressed as mean ± SEM, n = 5 for each time point and group. ** p < 0.01 – EO group versus CG and Sham groups at 4th day after wounding; *** p < 0.001 – EO group versus CG and Sham groups 7 days after wounding. Two-way ANOVA. Scale bar: 200 μm.

Figure 4. An increased collagen fibers deposition was noticed in wounds of EO group. (a) Kinetics of the percentage of collagen fibers. (b) Representative photomicrographs of Gomori’s trichrome-stained sections at the 7th postoperative day. Morphologically, collagen fibers presented thicker and more organized in the EO group, as compared to CG and Sham groups. Data are expressed as mean ± SEM, n = 5 for each time point and group. ** p < 0.01 – EO group versus CG; ***p < 0.001 – EO group versus Sham at the 7th postoperative day, and *p < 0.05 – EO group versus CG and Sham at the 14th postoperative day. Two-way ANOVA. Scale bar: 200 μm.