Bioactivity-Guided Isolation of Stigmasterol from Bursera bipinnata Resin: Pharmacological Evidence for Wound-Healing...

Background/Objectives: Bursera bipinnata (DC.) Engl. resin (locally known as "copal blanco") is traditionally used in Mexican ethnomedicine to treat infected wounds and skin inflammation, but the bioactive constituents underlying these effects remain largely uncharacterized. This study aimed to identify the compounds responsible for the wound-healing properties of the resin through bioactivity-guided fractionation and to evaluate their anti-inflammatory and antibacterial activities as complementary mechanisms supporting tissue repair. Methods: Crude resin (1.2-5.0 mg/mL) was assayed for anti-inflammatory activity in the TPA-induced ear-edema model in BALB/c mice, for antibacterial activity (MIC) against six clinically relevant strains, and for wound-healing activity in a murine excisional model with pirfenidone (PFD) as the reference drug (n = 5 per group). Bioactivity-guided fractionation followed by spectroscopic elucidation (¹H- and ¹³C-NMR, IR, EI-MS) led to the isolation of five constituents. Stigmasterol, the most active compound, was subsequently evaluated in an LPS-induced systemic inflammation model (oral administration, 20 mg/kg/day × 3 days) to characterize its immunomodulatory profile (TNF-α, IL-1β, IL-6, IFN-γ, IL-10) and in the wound-healing model to quantify local IL-6, IL-10 and TGF-β1 in skin homogenates. Results: The crude resin (5.0 mg/mL) achieved 99.63% wound closure at day 12 and a 49.08% reduction in TPA-induced ear edema, comparable to indomethacin (55.76%). The resin displayed selective antibacterial activity against Streptococcus pyogenes (MIC 125 µg/mL) and Salmonella typhimurium (MIC 250 µg/mL). Bioactivity-guided fractionation yielded the phytosterol stigmasterol (1), three lupane-type triterpenoids (lupeol acetate (2), lupenone (3), 3-epilupeol (5)), and the sesquiterpenoid caryophyllene oxide (4). At an equimolar 1 µM concentration, stigmasterol (1) shortened the mean wound-healing time to 10.3 ± 0.4 days, comparable to pirfenidone, and was associated with attenuation of systemic TNF-α, IL-1β and IL-6 peaks and with sustained local IL-10 and TGF-β1 expression. Histological assessment confirmed accelerated re-epithelialization and improved collagen organization. The resin was non-irritant in the OECD 404 acute dermal test (Primary Irritation Index = 0.00). Conclusions: These findings provide pharmacological evidence supporting the traditional use of B. bipinnata resin for wound healing. Stigmasterol (1), together with the lupane-type triterpenoids lupenone (3) and 3-epilupeol (5), were identified as key bioactive constituents. The data are consistent with a coordinated immunomodulation, in which stigmasterol is associated with reduced systemic pro-inflammatory signalling and increased local IL-10/TGF-β1 expression, an interpretation that should be confirmed in chronic and impaired wound-healing models.