The Effect of Topical Administration of an Ointment Prepared From Trifolium repens Hydroethanolic Extract on the Acceleration of Excisional Cutaneous Wound Healing

Wound healing is a 4-stage process, including homeostasis, inflammation, proliferation, and remodeling/maturation. Macromolecules such as carbohydrates are used for energy supply during the wound healing process.^1,2 Carbohydrates are used both as a main source of energy and for collagen synthesis.³ Mast cells play important roles in synthesizing different growth factors and the angiogenesis process during the wound healing process. The literature has shown that mast cells increased the angiogenesis ratio and inhibited the inflammation stage.^4,5 Apoptosis is an important process for removing the inflammatory cells and debris in granulation tissues. Several factors, such as the B-cell lymphoma 2 (Bcl-2) gene, stimulate and/or inhibit apoptosis at different stages of wound healing.⁶Bcl-2 families are involved in the apoptosis process. It has been found that p53 can induce apoptosis in inflammatory cells and removed them.^7,8

Synthetic drugs are used in wound healing processes, but they can have limited use due to their side effects. Medicinal plants are extensively utilized for wound healing. The role of antioxidant agents in the wound healing process is well known.⁹ As antioxidant compounds are found in a variety of plant species, flavonoids have antioxidant properties similar to synthetic agents. These compounds not only act as antioxidants, but also participate in the synthesis of collagens, inhibition of inflammation, and angiogenesis.^10,11 The Trifolium (Fabaceae) genus comprises 300 species distributed in both hemispheres.^12,13

Historically, the Trifolium species was used as antispasmodic, anti-inflammatory agents for the treatment of chronic skin diseases and whooping cough.¹⁴Trifolium repens has high amounts of flavonoids, thus showing antioxidant activities.¹⁵ Safer than synthetic agents, natural agents may enhance wound healing with their antioxidant and anti-inflammatory properties. T repens is an antioxidant and anti-inflammatory compound that may ameliorate the wound healing process. To the authors’ knowledge, there has been no research investigating the effect of ointments prepared from T repens hydroethanolic extract (TRHE) on the wound healing process through assessment with p53, Bcl-2, and Bcl-2-like protein 4 (BAX) gene expression, carbohydrate uptake, granulation tissue formation, mast-cell infiltration; angiogenesis, and collagen synthesis in rats.

Preparation of the plant extract
T repens flowers were collected from Hamadan (Hamadan Province, Iran) and authenticated by an expert botanist in the Department of Botany Sciences, Agriculture and Natural Resources Research Center in Hamadan. A voucher specimen (ID: RRCH: 016) was deposited in the herbarium of the same center. The powdered flowers (200 g) were suspended in 500 mL of hydroethanolic solution at room temperature (25ºC) for 96 hours. The mixture was filtered by fine muslin cloth and filter paper (Whatman No 1), dried in an oven at 40ºC for 96 hours, and finally kept at -20ºC for future experiments.¹⁶

Determination of total phenol and flavonoid content
Total phenolic content was evaluated by the Folin-Ciocalteu method.¹⁷ Briefly, after mixing the sample with 0.2 mL of Folin-Ciocalteu reagent, 2 mL of water, and 1 mL of 15% sodium carbonate (Na₂CO₃), the mixture was incubated; absorbance was measured at 765 nm. Flavonoid content was assessed, as reported in the literature¹⁸; the content was reported as milligram of quercetin equivalents per gram of dry extract. The high-performance liquid chromatography (HPLC) analysis of the selected flavonoids was conducted using myricetin, kaempferol, quercetin, and rutin, as described by Tundis et al.¹⁹ 

Radical scavenging DPPH and ABTS activity assays
The 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) assays were employed to evaluate the radical scavenging activity. For this purpose, ABTS radical cation (ABTS^·+) solution was initially blended with potassium persulphate and placed in the dark for 12 hours prior to application. Ethanol was used to dilute the ABTS^·+ solution. The extract was added, and the absorption was measured. To evaluate the DPPH, a DPPH ethanol solution (1.0 × 10⁻⁴ M) was mixed with different concentrations of samples (10-100 µg/mL); the solution was then incubated for 30 minutes in the dark, and the absorbance was read at 517 nm. Ascorbic acid was applied as a positive control. 

In vitro cytotoxicity and cell proliferation study
Human fetal foreskin fibroblast (HFFF2) cell lines cultured in RPMI 1640 (Gibco, Invitrogen) were supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Invitrogen), 2 mg/mL sodium bicarbonate, 0.05 mg/mL penicillin G (Serva), and 0.08 mg/mL streptomycin (Merck); afterwards, the cell lines were incubated at 37ºC with humidified air containing 5% CO₂. The effect of the extract was determined by MTT assays at different times.²⁰ In brief, 5000 cells per well were primarily cultured in a 96-well plate, incubated at 37ºC, and treated with serial concentrations of (0–10 000 µg/mL) for 24, 48, and 72 hours in triplicates. Cells treated with 0 mg/mL extract and 200 µL culture medium were served as controls. 

Following incubation, the medium in all plate wells was replaced with a fresh medium, and the cells were left for 24 hours in an incubator. After removing the medium, 50 µL of 2 mg/mL MTT (Sigma-Aldrich) dissolved in phosphate-buffered solution was added to each well; next, the plate was covered with aluminum foil and incubated for 4.5 hours. After removing the contents of the wells, 200 µL of pure dimethyl sulfoxide was added to them; the absorbance of each well was immediately read at 570 nm by a microplate absorbance reader (EL×800; Bio-Tek Instruments, Inc). Cell viability was calculated as shown in Formula 1.

Circular excision wound model
All the procedures were approved by the Ethics Committee of the Faculty of Veterinary Medicine, Islamic Azad University, Urmia Branch, Urmia, Iran (No. IAUU, 1109). The animals (N = 96) were anesthetized using ketamine and xylazine hydrochloride. Following surgical preparation, a circular, surgical full-thickness wound with an area of 314 mm² was created on the anterior-dorsal side of each rat.²¹ Next, all rats were randomly divided into 4 main groups (n = 18), and each group was subdivided into 3 subgroups (n = 6), including controls (group I), treated with commercial base formulation containing 90% soft paraffin, 5% hard paraffin, and 5% lanolin.²¹ In groups II, III, and IV, 1.5% TRHE, 3% TRHE, and 6% TRHE were mixed with the base formulation (T repens), respectively.²¹ The ointments were topically applied on the wound area once daily (1 g/day) from wound induction up to its completion. Until complete epithelialization, all rats were monitored for any wound fluids and/or signs of infection and other abnormalities. The wound area was measured by promptly placing a transparent paper on the wound and tracing it. The area of this impression was calculated using a graph sheet. The wound area was measured at the beginning of the experiments and on days 3, 6, 9, 12, 15, 18, and 21 post wounding. The percentage of wound closure was specified by an initial and final area drawn on glass slides during the experiments, as described in Formula 2.

Histological analysis
Histological analyses were carried out as described by previous studies. ^21-23 The animals were euthanized at days 3, 7, 14, and 21 after wounding; afterwards, the full-thickness tissue samples of 1 mm to 2 mm from the surrounding normal skin fixed in neutral-buffered 10% formalin were prepared, processed, embedded with paraffin wax, sectioned at 5-µm thickness, and stained with Masson’s trichrome. After cutting, the surrounding part was removed, and the wound lacking a surrounding part was considered for analysis. The samples stained by periodic acid-Schiff (PAS) for intracytoplasmic carbohydrate and alkaline phosphatase staining were utilized for tissue inflammation.^21,23 The sections were studied by ordinary light microscope (Olympus CX31RBSF attached to a camera). Three parallel sections were obtained from each specimen. Tissue sections were stained using the Masson’s trichrome method, toluidine-blue staining, and immunohistochemical staining for collagen, mast-cell distribution, and angiogenesis, respectively. Inflammatory cell infiltration and fibroblast/fibrocyte proliferation (per 1 mm² of the wound area), edema, and collagen deposition were evaluated in each tissue section. Collagen deposition was assessed via Image Pro-Insight software (Media Cybernetics) (background intensity section). After that, edema was analyzed in all groups, as indicated in the literature.^21-23 All parameters were analyzed in each high-power field (HPF), and the epithelial thickness was determined by a morphometric lens (Olympus).Edema, collagen score, and PAS were blindly examined in 5 HPFs by 2 pathologists unaware of the design and experimentation programs of the study. 

RNA isolation and cDNA synthesis
Total RNA was extracted from the wound areas by the standard TRIZOL method.²⁴ About 50 mg to 100 mg of a tissue sample per animal in each group was homogenized in 1 mL of TRIZOL. The isolated RNA was stored at -70ºC, and its amount was specified by spectrophotometer (260 nm and 260/280=1.8-2.0). The cDNA was synthesized in 20 µL of reaction mixture containing 1 µg RNA, oligo (dT) primer (1 µL), 5×reaction buffer (4 µL), RNAse inhibitor (1 µl), 10 mM dNTP mix (2 µL), and M-MuLV Reverse Transcriptase (1 µL), as recommended by the manufacturer’s protocol (Fermentas, GmbH). The cycling protocol for 20 µL of reaction mix was 5 minutes at 65ºC. As elucidated by Manzuoerh et al,²⁴ the polymerase chain reaction (PCR) was performed in the same volume of PCR master mix, forward, reverse specific primers, cDNA, and nuclease-free water. Ultimately, the reaction products were separated on 1.5% agarose gel and visualized by ethidium bromide staining using Gel Doc 2000 (Bio-Rad). The primer sequences were Bcl-2, forward (5’-CTGGTGGACAACATCGCTCTG-3’) and reverse (5’-GGTCTGCTGACCTCATTGTC-3’); BAX, forward (5’-TTCATCCAGGATCGAGCAGA-3’) and reverse (5’-GCAAAGTAGAAGGCAACG-3’); p53, forward (5’-GAGGAGATGATGCTGCTGAG-3’) and reverse (5’-TGCTGCTGCTGCTATTACC-3’); and ’Actin, forward (5’-CTGACCGAGCGTGGCTACAG-3’) and reverse (5’-GGTGCTAGGAGCCAGGGCAG-3’). 

Statistical analysis
All analyses were performed by PASW version 18.0 with the data presented as mean ± standard deviation. The results were analyzed by a two-way analysis of variance, and the effects of time and treatments were assessed by Dunnett’s test. A value of P < .05 was considered to be statistically significant.

Antioxidant activity, total phenols, and flavonoid contents
The TRHE contained phenol and flavonoid contents of 83.2 mg chlorogenic acid per gram extract and 20.3 mg of quercetin equivalents per gram of extract, respectively. The data obtained from HPLC analyses showed TRHE contained high contents of rutin (44.7 mg/g dry extract) and quercetin (11.4 mg/g dry extract). However, concerning kaempferol and myricetin, the values were negligible (0.5 mg/g and 1.4 mg/g dry extract, respectively).

Antioxidant activity was investigated by ABTS and DPPH methods, with the highest potency belonging to TRHE with 50% inhibition (IC50) values of 23.1 µg/mL and 11.3 µg/mL, respectively. Regarding ascorbic acid, however, the values of ABTS and DPPH were 1.6 µg/mL and 5.1 µg/mL, respectively.

In vitro cytotoxicity and cell proliferation assay
The growth of HFFF2 fibroblast cell lines was assessed at different times and concentrations of TRHE; results showed the cell toxicity against the growth of HFFF2 fibroblast cells increased with the rise in the concentration of the extract. The extract with the highest concentration had the most cytotoxic activity (Figure 1). 

Wound contraction
Topical administration of different doses of T repens to the excised wound significantly increased the rate of wound contraction compared with the control animals (P < .05). All wounds treated with doses of T repens exhibited wound closure on day 7; however, the closure was more pronounced at a dose of 6% (P < .05). The data of days 7 to 14 indicated that animals treated with a 6% dose of T repens (P < .05) had higher wound contraction rates (98%) in comparison with doses of 1.5% (88%) and 3% (94%) as well as the control group (79%) on day 14 (Table 1). 

Intracytoplasmic carbohydrate storage
Histochemical PAS staining showed that the topical administration of T repens in a dose-dependent manner upregulated the intracytoplasmic carbohydrate storage (Table 2). The animals treated with a high dose (6%) of T repens exhibited intensive reaction for PAS staining compared with those treated with 1.5% and 3% in connective cells (particularly fibroblasts and fibrocytes) (P < .05). Light microscopic analyses of carbohydrate foci in epithelial cells in all groups showed that animals treated with T repens, in a dose-dependent manner, had a better reaction for PAS staining (Figure 2).

Mast-cell distribution
Special staining of mast cells showed the topical administration of T repens, especially at a dose of 6%, significantly increased the mast-cell infiltration compared with control animals on all days after wound induction (P < .05). The highest mast-cell infiltration was observed on day 7 post wounding (P < .05) (Figure 3). 

Collagen deposition, connective tissue cells, reepithelialization, and edema
Topical administration of T repens increased well-formed granulation tissues on day 3 post wound induction and reduced the infiltration of immune cells compared with control animals on day 7 after wounding (P < .05) (Figure 3). Inflammation and fibroblast and fibrocyte distribution were lower and higher in rats treated with 6% T repens, respectively (Figure 4, Figure 5). A higher collagen deposition (particularly in the deeper dermis) was observed in T repens-treated groups (Figure 4). The animals in the 6% T repens-treated group showed reepithelialization on day 7 following the induction of wound; however, thin epithelialization was detected in the control group on day 14 post wound induction (P < .05) (Figure 4). On days 14 and 21, the newly generated epithelium was significantly thicker in the treated animals compared with the control animals (P < .05). Topical administration of T repens at a 1.5% dose on day 7 and at 3% on day 14, completely prevented edema (P < .05) (Table 2). 

Angiogenesis
On day 3 post wounding, neovascularization significantly increased in the T repens-treated animals (P < .05), contrary to the control group. The highest angiogenesis ratio belonged to the animals treated with 6% T repens on day 7 (P < .05), whereas those treated with 1.5% and 3% T repens exhibited massive angiogenesis on day 14 after wounding (P < .05) (Figure 5).

Molecular results
Administration of T repens at all dose levels decreased p53 and BAX expressions compared with the control group (P < .05); however, the animals in the control group showed increased messenger RNA (mRNA) levels of p53 and BAX 7 days after wounding (P < .05). The mRNA level of Bcl-2 significantly increased in the animals treated with T repens compared with the control group (P < .05) (Figure 6).

Phytochemical chemicals, such as flavonoids and triterpenoids, have been reported to promote the wound healing process.^25,26 The present findings showed that rutin and quercetin were major compounds in TRHE, which have been reported as major compounds in T repens extract by Xiong et al.²⁷ Tundis et al¹⁹ showed TRHE had a DPPH radical scavenging ability with an IC50 value of 10.30 µg/mL. It can be stated that TRHE has a stronger antioxidant activity compared with ascorbic acid. Inflammation-exerted oxidative stress changes the physiologic activities and metabolism during the healing process.²⁸ In this regard, neovascularization is a defensive and physiologic structural procedure that reduces the free radicals and metabolite delivery of cells involved in the healing process.²⁹ The present biochemical analyses on the herbal composition of TRHE revealed that this plant contained high contents of phenols and flavonoids. The shortened wound healing process may be attributed to the phenol- and flavonoid-induced antioxidant properties. 

Based on the present results, administration of T repens upregulated the expression of Bcl-2 and downregulated the expression of p53 and BAX; nevertheless, the animals in the control group showed a lower Bcl-2 expression and higher p53 and BAX expressions on day 7. The administration of T repens initiated cellular proliferation. BAX and p53 support the genome and prevent damages in cells through distinguishing and stopping the cell cycle.²⁴ After wounding, p53 and BAX expressions increased and promoted the immune cells apoptosis, thereby initiating the elimination of the immune cells. Following this phase, Bcl-2 prevented apoptosis and triggered cellular proliferation.^24,30 As previously mentioned, the Bcl-2 family inhibits apoptosis.^31,32 The results of reverse transcription PCR showed the administration of T repens increased Bcl-2 expression and reduced the expressions of p53 and BAX. It can be concluded that T repens increased the cellular proliferation via upregulating the Bcl-2 expression and reducing p53 and BAX mRNAs. Another important factor for cellular proliferation is the elimination of the immune cells from granulation tissues. More than 70 years ago, Mancini³³ reported that faulted fibroblast numbers increased pathological injuries for granulation tissue, formation of hypertrophied scar tissues, and keloids. 

Mast cells play major roles in secreting fibroblast growth factor-2 and participating in collagen synthesis through increasing the physiologic interaction of fibroblasts in the wound healing process.^34,35 Mast cells secrete vascular growth factor, which stimulates the proliferation of endothelial cells and completes the upregulation of neovascularization in wound healing.^35,36 Even in the preliminary stages, downregulated inflammation associated with provoked angiogenesis promotes proliferative machinery, such as fibroblasts, fibrocytes physiologic, and/or compensatory functions.³⁷ The present findings showed the distribution of mast cells increased in T repens-treated animals. It can be stated that the topical administration of T repens promoted the wound healing process and the proliferation and maturation stages through provoking mast-cell proliferation and distribution and upregulating the neovascularization (Figure 7).

In the animals treated with 6% T repens, angiogenesis was upregulated and vascularization increased on day 3 following wound induction. Vascular distribution (per 1 mm² of the tissue) in 6% T repens-treated groups was 3 to 4 times higher than those of the control group on day 7. Flavonoid contents of T repens upregulated the cellular profiles. Higher intracytoplasmic carbohydrate ratio increased the fibroblasts and fibrocytes; mast-cell distribution per 1 mm² of the tissue increased in the animals treated with high doses (ie, 6%) of T repens. 

The intracytoplasmic carbohydrate ratio differs among various cell types under different injury conditions.^2,33,38 Carbohydrates are used for producing hyaluronic acid, proteoglycan, and epithelium of glycosaminoglycan and injured skin (particularly in stratum spinosum).^30,32,38 The results of staining carbohydrates (PAS staining) showed that the administration of 3% and 6% T repens increased the expression of PAS-positive compared with the control animals. Furthermore, the stratum spinosum of the epidemis increased the intracytoplasmic carbohydrate storage in the animals treated with 6% T repens. From the results of this study, it is plausible that treatment with T repens increased carbohydrate ratio in dermal and epidermal cells, supplied the energy required for cells, and acted as a substrate necessary for the synthesis of structural materials. 

This study was conducted on a rat model with no disease and with an acute wound. Application of these findings to clinical wounds requires significantly more steps of research, such as human study with western blotting. 

The results of the current study showed high flavonoid and phenol contents in TRHE reduced the inflammation-induced degenerative effects. Moreover, administration of T repens increased mast-cell distribution and intracytoplasmic carbohydrate ratio, promoted angiogenesis, and shortened the wound healing process. Natural agents are safer than synthetic ones and capable of treating acute wounds without infection. The extract is economical and can be easily prepared. T repens can be recommended as natural compounds for preparing an ointment for wound healing; however, the results are preliminary and based on a rat model with no disease and an acute wound. 

Authors: Seied Kiavash Habibi Zadeh, DVM¹; Mohammad-Reza Farahpour, DVM, DVSc²; and Hamed Hamishe Kar, PhD³

Affiliations: ¹Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran; ²Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University; and ³Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Correspondence: Mohammad-Reza Farahpour, DVM, DVSc, Associate Professor, Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, 57159-44867, Iran; mrf78s@gmail.com 

Disclosure: The authors disclose no financial or other conflicts of interest.