Fresh C. asiatica leaves were sourced from Chian Yai Subdistrict and Mae Chao Yu Hua Subdistrict, Nakhon Si Thammarat Province, Thailand. Herbarium voucher specimens were C. asiatica SMD 0324030901. They were deposited at Applied Thai Traditional Medicine, School of Medicine, Walailak University, Nakhon Si Thammarat, Thailand. The plant materials were dried using a hot air oven at 60 °C for 72 h. All dried herbs were ground into coarse powders. To obtain crude ethanolic extracts, pulverized herbs were macerated in 95% ethanol (1:10 w/v) for 7 days with occasional stirring. The extract was filtered and concentrated under reduced pressure using a rotary evaporator. The final ethanolic extract was stored at 4 °C for further use.

Liposomes were prepared using the thin-film hydration method. Briefly, phosphatidylcholine (30 mg) and cholesterol (10 mg) were dissolved in chloroform (10 mL) in a round-bottom flask, followed by the addition of the ethanolic extract of Centella asiatica (10 mg). The solvent mixture was evaporated under reduced pressure using a rotary evaporator at 40 °C and 150 rpm to form a uniform thin lipid film along the inner wall of the flask. The resulting lipid film was hydrated with 5 mL of phosphate-buffered saline (PBS, pH 7.4) and vortexed for 5 min to obtain a milky liposomal suspension.

The liposomal dispersion was subsequently extruded through a 100 nm polycarbonate membrane using a mini-extruder (Avanti Polar Lipids, USA) to obtain vesicles with a uniform size distribution. Unencapsulated extract was removed by centrifugation at 15,000 × g for 30 min at 4 °C, and the resulting liposomal pellet was re-dispersed in PBS and stored at 4 °C until further use.

The UV–visible absorption spectra (200–800 nm) of both the ethanolic extract and the liposomal formulation were recorded to identify characteristic absorption peaks of phytoconstituents and to quantify extract content using pre-established calibration curves of reference standards.

The hydrodynamic particle size and polydispersity index (PDI) of the liposomes were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK) at 25 °C. Measurements were performed in triplicate, and representative size distribution profiles are presented.

The zeta potential of the liposomal formulation was measured by electrophoretic light scattering using the same Zetasizer Nano ZS instrument. Samples were appropriately diluted with deionized water to avoid multiple scattering effects and analyzed at 25 °C. Zeta potential values were calculated from electrophoretic mobility using the Smoluchowski approximation. Measurements were conducted in triplicate, and results are reported as the observed measurement range (−35 to −25 mV), indicating moderate colloidal stability of the liposomal formulation (36).

Encapsulation efficiency (EE%) of the liposome-encapsulated C. asiatica ethanolic extract was determined by quantifying the amount of extract entrapped within the liposomal vesicles. Briefly, the liposomal dispersion was centrifuged at 15,000 × g for 30 min at 4 °C to separate unencapsulated (free) extract from the liposome-associated fraction. The supernatant containing the free extract was carefully collected, and its absorbance was measured using a UV–visible spectrophotometer at the characteristic wavelength of the extract, based on previously established calibration curves.Encapsulationefficiencywascalculatedusingthefollowingequation:EE(%)=(Totalextract−Freeextract/Totalextract)×100

Mouse macrophage cells (RAW 264.7, ATCC TIB-71) were used for anti-inflammatory assays, while normal human dermal fibroblasts (NHDF; ATCC PCS-201-012™) were employed to model skin wound healing. Both cell lines were obtained from the Faculty of Medicine, Chulalongkorn University (Bangkok, Thailand). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and maintained at 37 °C in a humidified incubator with 5% CO₂. The study included four treatment groups: Negative control (Blank Liposome): Liposomes prepared without C. asiatica extract. Positive Control (Vitamin E). LEC Group (Liposome-Encapsulated Extract): Liposome-encapsulated C. asiatica ethanolic extract. EE Group (Ethanolic Extract): Crude ethanolic extract of C. asiatica.

RAW 264.7 cells were pre-treated with LPS (1 μg/mL) for 24 h to induce an inflammatory response. Cells were then treated with different concentrations of liposome-encapsulated C. asiatica ethanolic extract, blank liposome base, ethanolic extract, or vitamin E. The levels of pro-inflammatory cytokines (TNF-α, IL-1β) in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA, BD Biosciences, San Jose, CA) after 24 h of treatment (17, 18).

Healthy male Wistar albino rats (150–200 g) were housed in standard stainless-steel cages under controlled environmental conditions (12 h light/dark cycle, 21 ± 1 °C, 50 ± 10% relative humidity) with free access to food and water. After a 7-day acclimatization period, twenty animals were randomly assigned to experimental groups. All animal procedures complied with institutional and national ethical guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC), Prince of Songkla University, Thailand (Ethics Approval No. AR029/2025).

DLS analysis of the selected LEC formulation (100 µg/mL) demonstrated nanosized vesicles with a mean hydrodynamic diameter of 120.6 ± 9.4 nm and a low polydispersity index (PDI) of 0.18 ± 0.03 (Figure 1A), indicating a narrow size distribution and good formulation homogeneity. The zeta potential of this formulation ranged between −35 and −25 mV, suggesting sufficient surface charge to ensure colloidal stability and minimize vesicle aggregation during storage (Figure 1C). Encapsulation efficiency (EE%) was evaluated across the tested extract concentration range (6.5–100 µg/mL), reaching a maximum EE of 82.5 ± 2.3% (Figure 1B). The high encapsulation efficiency indicates favorable interactions between the bioactive phytoconstituents and the lipid matrix, supporting the suitability of the liposomal system for enhanced delivery and subsequent in vitro and in vivo applications.

The liposome-encapsulated C. asiatica extract demonstrated significant inhibition of pro-inflammatory cytokines compared to the blank liposome base. The reduction in TNF-α and IL-1β levels was more pronounced than with the crude ethanolic extract, indicating superior anti-inflammatory efficacy of the liposomal formulation (Figures 2A,B). The results of the study demonstrate that liposome-encapsulated C. asiatica extract (LEC) significantly reduced the levels of pro-inflammatory cytokines TNF-α and IL-1β in a dose-dependent manner (p < 0.05) compared to both the ethanolic extract (EE) and the blank liposome base. At lower concentrations (6.5–12.5 µg/mL), LEC achieved a substantial reduction in cytokine levels, indicating enhanced anti-inflammatory efficacy even at minimal doses. For example, at 12.5 µg/mL, LEC reduced TNF-α and IL-1β levels by 66.8 ± 3.4% and 69.5 ± 3.5%, respectively (Figures 1, 2), which was significantly greater than the reductions observed in the EE group at the same concentration (52.8 ± 3.0% for TNF-α and 56.4 ± 3.1% for IL-1β; p < 0.05). At higher concentrations (50–100 µg/mL), the efficacy of LEC was further amplified, with reductions of 80.4 ± 4.0% and 85.7 ± 4.3% for TNF-α at 50 and 100 µg/mL, respectively. These values were superior to the positive control group (vitamin E), which showed 83.2 ± 3.4% and 88.5 ± 3.5% reductions at the same concentrations. This suggests that LEC could serve as a more effective alternative or complementary anti-inflammatory treatment. The ethanolic extract (EE) showed moderate efficacy but was consistently less effective than LEC across all tested concentrations. For instance, at 100 µg/mL, EE reduced TNF-α levels by 72.5 ± 3.6%, markedly lower than LEC at the same concentration (85.7 ± 4.3%). The blank liposome base exhibited negligible anti-inflammatory activity, with cytokine reductions below 10% in all cases, confirming that the observed effects were primarily due to the active compounds in C. asiatica (Figures 2A,B).

LEC exhibited superior cell viability across all tested concentrations, demonstrating minimal cytotoxicity compared to the ethanolic extract (EE) and positive control (vitamin E). At the lowest concentration (6.5 µg/mL), LEC maintained cell viability at 99.2 ± 1.4%, slightly higher than EE (97.4 ± 1.9%) and significantly better than vitamin E (85.3 ± 2.8%). Even at the highest concentration (100 µg/mL), LEC displayed a viability of 89.2 ± 3.6%, indicating its safety and biocompatibility for wound healing applications. In contrast, EE showed a progressive decline in cell viability at higher concentrations, with a viability of 81.2 ± 3.5% at 100 µg/mL. This decline suggests potential cytotoxic effects at higher doses. Vitamin E exhibited consistent viability (85%–93%) across all concentrations but was slightly less effective than LEC, particularly at lower concentrations. The blank liposome control exhibited high viability (98.7 ± 1.5%), confirming that the liposome base itself is non-toxic (Figure 3A).

Fibroblast migration, a critical factor for wound healing, was significantly enhanced by LEC, particularly at concentrations between 12.5–50 µg/mL. At 12.5 µg/mL, LEC achieved a migration rate of 72.8 ± 4.2%, surpassing both EE (42.2 ± 2.8%) and vitamin E (65.3 ± 3.5%) at the same concentration. The highest migration rate for LEC was observed at 100 µg/mL (87.2 ± 3.7%), which outperformed all other groups, including vitamin E (72.8 ± 3.8%). The ethanolic extract (EE) showed moderate efficacy, with migration rates peaking at 25 µg/mL (60.5 ± 3.6%) but declining at higher concentrations. This suggests that while EE can stimulate fibroblast activity, its efficacy is dose-limited compared to LEC. The blank liposome control showed limited fibroblast migration (43.5 ± 2.6%), indicating that the enhanced migration observed in LEC was due to the active extract encapsulated in the liposomes (Figure 3B).

In the excision wound model, treatment with the liposome-encapsulated C. asiatica extract (LEC) gel exhibited a pronounced wound-healing effect compared with the control groups. By Day-2 post-treatment, animals receiving LEC showed a clear reduction in wound area, with the degree of wound closure closely approaching that of the vitamin E-treated group by Day-4. In contrast, wounds in the blank liposome and normal saline control exhibited minimal contraction during the same period (Figure 4).

Quantitative analysis of wound diameter and area confirmed that the LEC-treated group demonstrated a significantly higher percentage of wound contraction than the normal saline group throughout the 12-day observation period (p < 0.05; Table 1 and Supplementary Table S1). The rate of wound closure in the LEC group was comparable to that of the vitamin E-treated standard group from Day-4 onward, indicating that the liposomal formulation achieved early and sustained wound-healing efficacy. By Day-8, wounds in the control groups remained noticeably larger, whereas those treated with LEC or vitamin E had nearly complete epithelial coverage. These findings collectively suggest that topical application of LEC significantly accelerates wound contraction and promotes faster tissue regeneration, comparable to standard vitamin E treatment.

Histological examination of excised wound tissues on Day-12 revealed clear treatment-dependent differences in the progression of wound healing (Figures 5, 7B). Wounds treated with liposome-encapsulated C. asiatica extract (LEC) exhibited the most advanced histological features of tissue repair. The epidermis was completely re-epithelialized, forming a continuous and well-organized epithelial layer, which was reflected by the highest re-epithelialization score (2.6 ± 0.5). The underlying dermis showed dense fibroblast-rich granulation tissue and well-developed collagen architecture, accompanied by prominent neovascularization, indicating active tissue remodeling and angiogenesis (Figure 5A).

In the vitamin E treated group, wound healing was moderately advanced. The epidermis was largely continuous, although less uniform than in the LEC group, corresponding to an intermediate re-epithelialization score (1.8 ± 0.8). The dermis contained numerous fibroblasts and newly formed blood vessels, with moderately dense collagen deposition, suggesting ongoing granulation tissue formation and angiogenesis (Figure 5B). Semi-quantitative analysis confirmed intermediate fibroblast/granulation and neovascularization scores (1.6 ± 0.5 and 2.0 ± 0.7, respectively). In contrast, the blank liposome and normal saline groups exhibited delayed and less organized wound repair. These groups showed incomplete and fragmented epidermal coverage, sparse fibroblast proliferation, and limited collagen deposition, accompanied by reduced neovascularization (Figures 5C,D). Correspondingly, the re-epithelialization, fibroblast/granulation, and neovascularization scores were significantly lower in these control groups compared with the LEC-treated wounds (Figure 7B). Overall, the semi-quantitative histological scoring corroborates the qualitative microscopic observations and macroscopic wound closure data, demonstrating that liposomal encapsulation of C. asiatica extract significantly enhances epidermal regeneration, granulation tissue formation, and angiogenesis, thereby accelerating the wound-healing process.

On Day 12 post-treatment, Masson's trichrome staining was performed to evaluate collagen deposition and dermal remodeling in excised wound tissues (Figure 6). Clear treatment-dependent differences in both the extent and organization of collagen fibers were observed among the experimental groups. Wounds treated with liposome encapsulated C. asiatica extract (LEC) exhibited the most prominent collagen deposition. The dermis contained densely packed, compact, green-stained collagen fibers that were well aligned and oriented parallel to the epidermal surface, indicating advanced extracellular matrix remodeling. The regenerated epidermis was continuous and well stratified, consistent with near-complete re-epithelialization (Figure 6A).

In the vitamin E treated group, collagen deposition was also evident, with moderately dense and organized collagen fibers distributed throughout the dermis. Although collagen alignment was slightly less compact than that observed in the LEC group, the dermal architecture was largely preserved, with visible adnexal structures such as hair follicles and sebaceous glands. The epidermis appeared intact and stratified, reflecting an ongoing but less advanced remodeling phase compared with LEC treatment (Figure 6B). In contrast, the blank liposome group showed sparse and disorganized collagen fibers that were thin, irregularly oriented, and unevenly distributed within the dermis. The overall tissue architecture appeared disrupted, and epidermal regeneration was incomplete, indicating delayed wound repair (Figure 6C). The normal saline treated group demonstrated the poorest healing response, characterized by minimal collagen deposition with scattered green stained fibers, poor dermal organization, and a thin, fragmented epidermis (Figure 6D).

Quantitative morphometric analysis of Masson's trichrome-stained sections confirmed these observations (Figure 7A). The collagen area fraction was significantly higher in the LEC-treated group compared with the normal saline group (p < 0.01). Both the blank liposome and vitamin E groups also showed significantly increased collagen content relative to normal saline (p < 0.05), although the magnitude of collagen deposition was lower than that observed in the LEC group. Collectively, these findings demonstrate that LEC treatment markedly enhances collagen deposition, dermal organization, and epidermal regeneration, supporting accelerated wound remodeling compared with control and reference treatments.