During the inflammatory phase, polymorphonuclear neutrophils (PMNs) and macrophages infiltrate the wound site to eliminate debris and pathogens. Angiogenesis simultaneously ensures nutrient and oxygen supply to regenerating cells^4,5. In the proliferative phase, fibroblasts form granulation tissue composed of a provisional extracellular matrix rich in collagen, fibronectin, and hyaluronic acid^6,7. This stage plays a pivotal role in re-establishing structural stability, while the remodeling phase strengthens the extracellular matrix and restores functional integrity⁸.

Historically, medicinal plants and herbal preparations have been widely used in wound care, often based on empirical knowledge rather than validated scientific evidence^9,10. Despite this, herbal agents continue to attract attention due to their accessibility, affordability, and potential clinical efficacy¹¹.

C. officinalis (marigold) has traditionally been employed in Mediterranean cultures for the treatment of skin lesions¹². Its wound-healing potential is attributed primarily to anti-inflammatory activity. Phytochemical studies have identified a wide variety of active compounds, including flavonoids, coumarins, steroids, terpenoids, carbohydrates, lipids, tocopherols, quinones, carotenoids, essential oils, fatty acids, and minerals^13-15.

Based on these properties, the present study aimed to investigate the histopathological and biochemical effects of C. officinalis on wound healing and epithelialization in a rat model.

Laboratory Animals: Twenty-four male Wistar albino rats (200-240 g) were obtained from the Experimental Animal Center of Yüzüncü Yıl University. Animals were housed under standard laboratory conditions [25±2°C, relative humidity (50±15%), 12-h light/dark cycle] in individual wire-bottom cages with free access to food and water. Veterinary supervision was maintained throughout the study.

Plant Material and Extract Preparation: C. officinalis flowers were purchased from a local herbal store in Van, Turkey. The flowers were shade-dried, ground into powder, and 100 g of the material was extracted with 96% (v/v) ethanol using a Soxhlet apparatus. The extract was concentrated under reduced pressure at 40-45 °C using a rotary evaporator. A wound-healing ointment containing 5% (w/w) C. officinalis extract in glycerin was prepared and stored in amber-colored glass bottles until use.

Experimental Design and Wound Model: Rats were randomly divided into three groups (n = 8 per group):

Group 1 (Control): Received glycerin ointment only,

Group 2 (Reference-Centella asiatica): Treated with Madecassol® ointment.

Group 3 (C. officinalis): Treated with C. officinalis ointment.

To evaluate the wound healing potential of C. a officinalis extract, a topical pomade containing 1% Centella asiatica extract (Madecassol®; Bayer Türk Kimya San. Ltd. Şti., İstanbul, Türkiye) was employed as the positive control group. This topical preparation was utilized as a reference standard in the study due to the well-documented efficacy of C. asiatica on collagen synthesis and epithelialization.

Anesthesia was induced via intraperitoneal injection of ketamine hydrochloride (50 mg/kg body weight) and xylazine hydrochloride (10 mg/kg body weight). The dorsal region was shaved, depilated, and disinfected with antiseptic. A full-thickness circular excision wound (8 mm diameter) was created in the interscapular region using a sterile biopsy punch, extending into the subcutaneous tissue (Figure 1).

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Figure 1: Experimental design and procedure. A. Skin surface prior to wound creation. B. Postoperative topical treatment application. C. Measurement of wound area.

Treatments were applied topically once daily at 24-hour intervals, beginning immediately after wound induction and continuing until complete healing. Wound areas were measured on days 0, 4, 7, 12, and 15 by tracing margins onto millimeter graph paper, with surface areas calculated using AutoCAD software. Standardized digital photographs were taken at a fixed distance of 15 cm perpendicular to the wound surface. The animals were monitored clinically daily.

On days 7 and 15, four rats from each group were euthanized under anesthesia. Blood and wound tissues were collected for histopathological and biochemical analyses.

Histopathological Examination: Full-thickness wound tissues were excised, fixed in 10% neutral-buffered formalin for 48 h, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. Serial sections of 5 µm were prepared using a microtome and stained with hematoxylin and eosin (H&E). Sections were examined under light microscopy, and photomicrographs were obtained.

Histopathological parameters were semi-quantitatively scored as absent (-), mild (+), moderate (++), or severe (+++) for epidermal and dermal remodeling. Parameters included re-epithelialization, ulceration, fibroblast proliferation, mononuclear/polymorphonuclear cell infiltration, neovascularization, and collagen deposition. Based on these parameters, wounds were classified into inflammatory, proliferative, or remodeling phases¹⁶.

Biochemical Analysis
Lipid peroxidation: Malondialdehyde (MDA) concentrations were determined according to Jain et al.¹⁷ based on thiobarbituric acid reactivity.

Antioxidant substances and enzymes: Glutathione (GSH) levels were measured by the method of Beutler et al.¹⁸. Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined spectrophotometrically at 505 nm and 37 °C based on inhibition of formazan dye formation¹⁹. Catalase (CAT, EC 1.11.1.6) activity was determined using the method of Aebi²⁰

Serum biochemistry: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), total protein (TP), creatine kinase (CK), iron (Fe), glucose (GLU), albumin (ALB), phosphorus (P), magnesium (Mg), calcium (Ca), and urea were measured using an automated analyzer (BM/HITACHI-911) with commercial kits (DPC; Diagnostic Products Corporation, USA).

Statistical Analysis: All data were presented as means ± standard deviations. The statistical analyses were performed using the Minitab 13 for Windows. Normality and homogeneity of variance were assessed using the Shapiro-Wilk and Levene's tests, respectively. Differences among groups were analyzed using one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. A p value ≤0.05 was considered statistically significant. The Kruskal-Wallis test was used as a nonparametric alternative to evaluate histopathological scores.

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Figure 2: Representative macroscopic images of wound healing in the Control, C. asiatica, and C. officinalis groups on days 7 and 15. (A-C) Day 7; (D-F) Day 15

Macroscopic Findings: Macroscopic evaluation on days 0, 4, 7, 12, and 15 revealed distinct differences in wound morphology among groups. On day 4, control wounds exhibited irregular scab formation with heterogeneous crusting and localized exudation, whereas both treatment groups showed compact, homogeneous scabs. By day 7, control wounds still demonstrated irregular scabbing and persistent inflammation, while treatment groups displayed organized wound edges and accelerated epithelialization. On day 12, healing in controls was delayed, with incomplete scab loss and persistent fissures, whereas treatment groups showed advanced contraction, granulation tissue replacement, and near-complete scab removal. By day 15, residual scabs and scarring persisted in controls, while both treatment groups demonstrated nearly complete closure, minimal scarring, and uniform epithelial renewal. Representative macroscopic images are shown in Figure 3(A-F).

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Figure 3: Microscopic appearance of wound healing on days 7 and 15. H&E. A. Control group, day 7. B. C. asiatica group, day 7. C. C. officinalis group, day 7. D. Control group, day 15. E. C. asiatica group, day 15. F. C. officinalis group, day 15. Abbreviations: S: Scab, Re: Re-epithelization, nv: neovascularization, stars: exudate, c: connective tissue

Microscopic Findings: Figure 4 presents the microscopic findings according to the groups on the 7 (Figure 4A-C) and 15 (Figure 4D-F) days for wound healing process and healing phases. Histopathological analysis revealed clear differences in wound healing progression. In the control group, extensive scab formation (+++), moderate ulceration (++), absent or minimal re-epithelialization (-/+), weak fibroblast proliferation (+), irregular collagen deposition (-/+), and marked inflammatory cell infiltration (MNC ++/+++, PMN +++) were noted. Neovascularization was limited (-/+), and the healing phases remained insufficient (Figure 4A and D). In contrast, C. asiatica treatment resulted in limited scabbing (++), absence of ulceration (-), advanced re-epithelialization (+/++), pronounced fibroblast proliferation (++), and organized collagen deposition (++). Inflammatory infiltration was reduced, neovascularization was moderate (+/++), and proliferative/remodeling phases were prominent (++/+++) (Figure 4 B and E). The C. officinalis group showed a comparable profile: reduced scabbing (++), minimal ulceration (-/+), advanced re-epithelialization (+), marked fibroblast proliferation (++), regular collagen deposition (++), low inflammatory infiltration, and enhanced neovascularization (++). Both treatments facilitated efficient progression through healing phases, contrasting with the inflammation-dominant control group (Figure 4C and F). Quantitative histopathological scores are presented in Table 1.

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Figure 4: Histopathological evaluation of wound healing and epidermal/dermal remodeling in control, C. asiatica, and C. officinalis-treated rats on days 7 and 15. Hematoxylin and eosin (H&E) staining. A.Control group, day 7. B. C. asiatica group, day 7. C. C. officinalis group, day 7. D. Control group, day 15. E. C. asiatica group, day 15. F. C. officinalis group, day 15.

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Table 1: Histopathological evaluation of wound healing parameters in experimental groups

Lipid Peroxidation and Antioxidant Parameters: Control group wounds exhibited significantly elevated malondialdehyde (MDA) levels. Both C. asiatica and C. officinalis significantly reduced MDA concentrations (p≤ 0.05). Endogenous antioxidant defense markers were also improved: glutathione (GSH) levels, diminished in controls, were significantly increased in both treatment groups; superoxide dismutase (SOD) activity was higher, most prominently in the C. asiatica group; and catalase (CAT) activity, reduced in controls, was significantly elevated in the treatment groups, with a more robust effect in C. asiatica. These results indicate that both treatments attenuated lipid peroxidation and enhanced antioxidant defense, contributing to accelerated repair. Data are summarized in Table 2.

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Table 2: The effect of C. officinalis on MDA, GSH contents and antioxidant enzyme activities in wound healing tissue

Biochemical Findings: Biochemical assessment revealed significant group differences, particularly in hepatic enzyme activity and protein metabolism. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were elevated in controls but significantly reduced in both treatment groups (p≤0.05). Gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), urea, calcium, magnesium, phosphorus, creatine kinase (CK), glucose, and iron levels showed no significant variation (p>0.05). Total protein and albumin levels were significantly increased in treatment groups (p≤0.05), reflecting improved protein synthesis. Collectively, these results suggest hepatoprotective effects and improved protein homeostasis by both interventions, without adverse effects on glucose or mineral balance. Biochemical data are summarized in Table 3.

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Table 3: The effect of C. officinalis on some biochemical parameters in wound healing model in rats

In the present study, both C. asiatica and C.officinalis significantly enhanced wound contraction and closure compared with the control group, with measurable benefits evident from day 4 onward. Notably, C. asiatica demonstrated slightly earlier effects, whereas C. officinalis exhibited sustained healing throughout the observation period. The observed improvements are likely mediated by the plant’s bioactive constituents, including flavonoids, triterpenes, and saponins, which exert antimicrobial, anti-inflammatory, and antioxidant effects¹². Our findings are consistent with previous studies demonstrating the efficacy of C. officinalis and other phytotherapeutic agents. Ozturan and Akin²⁵ reported that C. officinalis extract shortened epithelialization time, reduced inflammation, and promoted fibroblast proliferation and collagen synthesis. Similarly, Parente et al.²⁶ demonstrated that C. officinalis positively influenced the proliferative phase and wound closure, likely due to its antimicrobial and reparative properties. In our study, by day 12, wounds in both treatment groups showed substantial closure, while those in the control group remained larger. By day 15, wounds in the treatment groups were nearly completely healed, consistent with the findings of Ejiohuo et al.¹² and Preethi and Kuttan^27, who attributed the plant’s wound-healing activity to bioactive compounds such as flavonoids, triterpenes, and saponins that mitigate oxidative stress by scavenging free radicals. These mechanisms collectively suggest that C. officinalis accelerates wound healing by modulating fibroblast function, extracellular matrix deposition, and angiogenesis. Taken together, C.officinalis demonstrated wound-healing effects comparable to C. asiatica, significantly reducing wound size and promoting tissue regeneration. While C. asiatica remains a well-established pharmaceutical standard, C. officinalis exhibited comparable efficacy despite being a botanical preparation, supporting its potential use as a complementary or alternative therapy for wound management.

The cellular and extracellular matrix dynamics within the epidermal and dermal layers are influenced by both local and systemic factors. In the present study, topical application of C. asiatica and C. officinalis extract led to marked histopathological improvements compared with the control group, reflecting their beneficial effects across multiple phases of tissue repair. Scab formation was notably more evident in controls but reduced in treated groups, indicating accelerated healing and earlier resolution of the wound surface³. Consistent with earlier findings, such rapid scab detachment corresponds to enhanced re-epithelialization and fibroblast proliferation (28, 29). Ulceration, which was moderate in the control animals, was absent or minimal in treated groups, supporting more effective epithelial restoration. Re-epithelialization was limited in the control group but well advanced in both C. asiatica and C. officinalis-treated rats, confirming their stimulatory roles in epidermal regeneration. These outcomes align with previous evidence that C. asiatica triterpenoids enhance fibroblast activity and collagen synthesis^30,31, while flavonoids in C. officinalis facilitate epithelialization and wound contraction²⁶. Accordingly, fibroblast proliferation and collagen deposition were significantly greater in the treatment groups, indicating enhanced extracellular matrix remodeling³². Inflammatory cell infiltration, which was intense in the control wounds, was markedly reduced in the treated groups, consistent with the documented anti-inflammatory properties of both C. asiatica and C. officinalis^27,33. Furthermore, angiogenesis was substantially increased following treatment, ensuring better vascularization of the healing tissue and supporting previous reports of the pro-angiogenic activity of C. officinalis³⁴. Overall, these findings demonstrate that both C. asiatica and C. officinalis significantly enhance wound repair by promoting re-epithelialization, fibroblast proliferation, collagen synthesis, and angiogenesis while attenuating inflammatory responses.

Oxidative stress is a major determinant of wound healing, as excessive reactive oxygen species (ROS) impair cellular proliferation, angiogenesis, and extracellular matrix remodeling, ultimately delaying tissue repair³⁵. In this study, significant intergroup differences were observed in both lipid peroxidation and antioxidant parameters, emphasizing the importance of redox balance in cutaneous regeneration. MDA, a key marker of lipid peroxidation and oxidative injury, was markedly elevated in controls, indicating increased oxidative stress and delayed healing. Conversely, C. asiatica and C. officinalis treatments significantly reduced MDA levels, consistent with previous reports on their antioxidative and wound-healing activities²⁶. GSH, a critical intracellular antioxidant maintaining redox homeostasis, was significantly elevated in both treatment groups, suggesting enhanced endogenous antioxidant capacity. Similar upregulation of GSH has been linked to triterpenoid components of C. asiatica and phenolic compounds in C. officinalis²⁷. SOD, responsible for converting superoxide radicals into less reactive species^35, exhibited higher activity in C. asiatica treated rats and moderate elevation in the C. officinalis group, supporting an improved oxidative environment favorable for tissue repair³⁶. CAT, which decomposes hydrogen peroxide into water and oxygen, followed a similar trend, with maximal activity in C. asiatica, intermediate in C. officinalis, and minimal in controls. Enhanced SOD and CAT activity reflects strengthened enzymatic defenses and reduced oxidative burden, thereby facilitating re-epithelialization and collagen synthesis³³. Collectively, these findings demonstrate that both C. asiatica and C. officinalis exert potent antioxidant effects by decreasing lipid peroxidation and enhancing enzymatic (SOD, CAT) and non-enzymatic (GSH) defenses. Although C. asiatica showed slightly stronger activity, C. officinalis exhibited robust antioxidant potential, validating its role as an effective phytotherapeutic adjunct in wound management. The biochemical improvements observed likely underlie the histopathological outcomes, linking enhanced oxidative balance to accelerated tissue regeneration.

In this study, systemic biochemical parameters were analyzed to evaluate the effects of C. asiatica and C. officinalis on experimentally induced skin wounds. Significant group differences were observed in ALT, AST, total protein, albumin, while GGT, ALP, urea, calcium, magnesium, phosphorus, CK, glucose, and iron showed no marked variation. The reductions in ALT and AST levels in both treatment groups suggest attenuation of hepatic enzyme activity, indicative of reduced hepatocellular stress. This hepatoprotective effect may result from decreased oxidative damage, consistent with previous findings that antioxidant therapies normalize elevated liver enzyme levels following tissue injury^7,37. The improvement observed in the C. officinalis group likely stems from its high flavonoid content and free radical–scavenging capacity²⁷. Increases in total protein and albumin levels further indicate enhanced protein synthesis and improved metabolic support for wound repair. As albumin contributes to cellular proliferation, collagen formation, and tissue remodeling^28, these changes reflect stimulation of regenerative processes, aligning with the reported immunomodulatory and reparative actions of C. officinalis²⁶. No significant differences were noted in other biochemical markers, indicating systemic stability during the healing process. Collectively, these findings demonstrate that both C. asiatica and C. officinalis modulate hepatic enzyme activity, enhance protein metabolism, and protect against tissue damage, thereby supporting both local and systemic aspects of wound healing.

In conclusion, C. officinalis exhibited significant wound-healing potential by promoting cellular proliferation, enhancing extracellular matrix formation, and facilitating organized tissue remodeling. Moreover, it improved key biochemical and histological parameters by mitigating oxidative stress and restoring redox balance, thereby creating a microenvironment conducive to effective tissue repair. These findings provide strong scientific support for its traditional medicinal application and underscore its promise as a safe, accessible, and effective phytotherapeutic agent for enhancing cutaneous wound healing in clinical settings.

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