Wound Healing Potential of Herbal Hydrogel Formulations of Cedrus brevifolia Extracts in Mice

Abstract

Wound healing stands as a paramount therapeutic pursuit, imposing significant challenges on healthcare, particularly for vulnerable populations. Cedrus brevifolia, a species endemic to Cyprus, thrives in the Tripylos region, commonly known as Cedar Valley, within the Paphos forest. Despite its endemism, this species exhibits negligible genetic divergence from its Mediterranean related species. This study aims to investigate the potential of C. brevifolia resin and bark extracts in promoting wound healing in a mouse model. Previous in vitro investigations have elucidated the antioxidant and anti-inflammatory potential of extracts and isolates derived from the title plant, warranting further exploration in an in vivo setting. This experimental design employed 40 male SKH-hr2 black and brown mice aged 2-4 months. Wounds measuring 1 cm² were meticulously induced in the anesthetized mice and the potential healing effect of the herbal hydrogel formulations was evaluated. The healing potential of the C. brevifolia extracts was rigorously assessed through the daily application of gel formulations containing resin concentrations of 5% and 10% w/w, alongside sapwood and heartwood extracts at concentrations of 0.5% and 1% w/w. The evaluation of the treatments encompassed a multifaceted approach, incorporating clinical observations, skin biophysical parameter assessments utilizing an Antera 3D camera, and FT-IR spectroscopy, in addition to histopathological examination. The chemical compositions were also investigated through NMR and bio-guided isolation. The most prominent herbal hydrogel preparation proved to be the 10% resin, followed by the sapwood at 1%. The chemical analysis unveiled abietic acid, manool, and lariciresinol derivatives that potentially contributed to the observed results. Bridging the gap between in vitro observations and in vivo outcomes attempts to shed light on the potential therapeutic benefits of C. brevifolia hydrogels in wound care.

Keywords: Cedrus brevifolia; FT-IR; NMR; SKH-hr2; hydrogel; in vivo; plant extract; wound healing.

Figure 1

Overlaid ¹H-NMR chemical fingerprints of Cedrus brevifolia resin (blue; in CDCl₃), sapwood H₂O (red; in MeOD), EtOH extracts (green; in MeOD), heartwood H₂O (purple; in MeOD), and EtOH extracts (yellow; in MeOD).

Figure 2

Photodocumentation throughout the experiment (days 1, 3, 5, 7, 9, 11, 13, and 14) displays the assessment of wound healing across eight groups: the control, the vehicle, and the groups that received a gel containing resin or C. brevifolia extracts (resin 5% and 10%, sapwood and heartwood extracts 0.5% and 1%, respectively).

Figure 3

Wound healing closure (%) in relation to time. Statistical significance: control vs. resin 10% and sapwood extract 1% (p < 0.01; **), heartwood extract 1% (p < 0.05), and vehicle vs. resin 10% and sapwood extract 1% (p < 0.05; *).

Figure 4

Skin sections at the end of the experimental procedure, stained with hematoxylin-eosin (100×). Representative annotation: the red ellipse depicts intense inflammatory elements; the back ellipse depicts parakeratosis; and the blue ellipse depicts epidermal hyperplasia.

Figure 5

TEWL, hydration, and hemoglobin measurements, taken before the wound infliction and on the last day of the experiment.

Figure 6

Represents a comparison of normalized and averaged FT-IR spectra on day 14 post-lesion for the healthy skin (blue), control group (untreated mice; red), vehicle control group (mice treated only with excipients; navy blue), 5% w/w (black) and 10% w/w (pink) resin gels, 0.5% w/w (purple) and 1% w/w (olive) sapwood extract gels, and 0.5% w/w (green) and 1% w/w (orange) heartwood extract gels. Changes in the band frequency, intensity, and shape were observed across the spectral regions 3700–3000 cm⁻¹, 3000–2850 cm⁻¹, and 1800–800 cm⁻¹.