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

Abstract and Introduction


Introduction: Natural agents with antioxidant and anti-inflammatory properties are safer than synthetic agents and may improve wound healing.

Objective: The purpose of this study is to evaluate the in vivo wound healing potential of an ointment prepared from Trifolium repens hydroethanolic extract (T repens) concerning excisional wounds in a rat model.

Materials and Methods. Seventy-two adult Wistar rats were divided into 4 groups: a control group and 3 groups of animals treated with 1.5% T repens, 3% T repens, and 6% T repens. A full-thickness wound with an area of 314 mm2 was created in each rat. To investigate the effect of T repens on wound healing, the wound area, histological analyses (eg, angiogenesis, fibroblast, fibrocyte, mast-cell distribution), intracytoplasmic carbohydrate storage, and B-cell lymphoma 2-like protein 4 (BAX), B-cell lymphoma 2 (Bcl-2), and p53 gene expression in the wound tissue were evaluated for 21 days. Antioxidant activity was further measured by 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) assays.

Results: The animals in the treated groups showed higher wound contraction ratios (P < .05), angiogenesis, fibroblast, fibrocyte, and mast-cell distribution and intracytoplasmic carbohydrate storage compared with the control group (P < .05). Moreover, the topical administration of T repens increased the messenger ribonucleic acid (mRNA) level of Bcl-2 and reduced the BAX and p53 mRNA levels (P < .05). These findings further revealed the strong antioxidant activity of T repens.

Conclusions: The topical administration of T repens accelerated wound healing by increasing angiogenesis; fibroblast, fibrocyte, and mast-cell distribution; intracytoplasmic carbohydrate storage; and modulation in genes involved in apoptosis in a rat model.


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.[6]Bcl-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.[14]Trifolium 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.