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

Disclosures

Wounds. 2020;32(9):253-261. 

In This Article

Results

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).

Figure 1.

Effect of Trifolium repens hydroethanolic extract on the proliferation of HFFF2 fibroblast cell lines. Cell viability percent was calculated with reference to the baseline control of 100%.
h: hour; T repens: Trifolium repens

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).

Figure 2.

Histological photomicrograph of the superficial dermis in the (A) control, (B) 1.5%, (C) 3%, and (D) 6% Trifolium repens-treated groups. Note the increased intracytoplasmic carbohydrate supplementation in connective tissue cells (arrows). (E–H) The epidermis is marked with lines, and the periodic acid-Schiff (PAS)-positive cells are shown with arrows. The epidermal cells exhibited a reduced intracytoplasmic carbohydrate ratio in the control animals compared with those in the T repens-treated groups. Groups are outlined as (E) control, (F) 1.5%, (G) 3%, and (H) 6% T repens-treated groups. PAS staining (A, B, C, D: magnification, 600x; and E, F, G, H: magnification, 400x).

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).

Figure 3.

Mast-cell distribution on day 7 post wounding in the (A) control, (B) 1.5%, (C) 3%, and (D) 6% Trifolium repens-treated groups. Note the intensive and significantly higher mast cells (arrows) infiltration in the 3% (C) and 6% (D) T repens-treated groups in comparison with the control (A) animals. Toluidine blue staining (magnification, 200x).

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 (Figures 4, 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).

Figure 4.

Histological photomicrograph of the skin on day 7 after wound induction in the (A) control, (B) 1.5%, (C) 3%, and (D) 6% Trifolium repens-treated groups. Note the dose-dependent increased angiogenesis (arrows) associated with good collagen deposition in the T repens-treated groups. Meanwhile, the angiogenesis and collagen synthesis was significantly lower in the control animals. The dermis of the (E) control, (F) 1.5%, (G) 3%, and (H) 6% T repens-treated groups. The dermis was completely mature in the 6% T repens-treated group compared with those of the other treated and control animals. Intensive collagen deposition is presented in the dermis of the 6% T repens-treated group. The papilla of the dermis was completely formed on day 21 (H; thick arrow).
Masson's trichrome stain (A–D: magnification, 600x) and (E–H: magnification, 400x).
EP: epidermis; D: dermis

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).

Figure 5.

Histological photomicrograph of the skin on day 7 post wound induction. Fluorescent staining for angiogenesis in the (A) control, (B) 1.5%, (C) 3%, and (D) 6% Trifolium repens-treated groups. Note the upregulation of angiogenesis in the (C) 3% and (D) 6% T repens-treated groups in comparison with the control group (A-D: magnification, 400x). (E) Mean distribution of vessels per mm2 of the tissue on days 3, 7, 14, and 21 post wounding.
All data are presented in the graph (E) as mean±standard deviation.
b, cRepresents significant differences versus a on the same day.

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).

Figure 6.

(A) The messenger RNA (mRNA) expression of B-cell lymphoma 2-like protein 4 (BAX), B-cell lymphoma 2 (Bcl-2), and p53 gene in different groups. Administration of Trifolium repens increased the mRNA expression of Bcl-2, p53, and BAX in comparison with the control group. The animals in the 6% T repens-treated group showed a significantly higher mRNA expression level based on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) intensity (P<.05) and a graph of (B) Bcl-2, p53, and BAX/GAPDH intensity. All data are expressed as mean±standard deviation.
a, b, c, dRepresent significant differences between the marked data.

Comments

3090D553-9492-4563-8681-AD288FA52ACE

processing....