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

Seied Kiavash Habibi Zadeh, DVM; Mohammad-Reza Farahpour, DVM, DVSc; Hamed Hamishe Kar, PhD


Wounds. 2020;32(9):253-261. 

In This Article

Materials and Methods

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 literature;[18] 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 ormula 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.