Topical Application of Premna integrifolia Linn on Skin Wound Injury in Rats Accelerates the Wound Healing Process: Evidence from In Vitro and In Vivo Experimental Models

Abstract

Background: When the skin and tissues within the body are injured, the healing process begins. Medicinal herbs have been used to cure wounds since time immemorial. The antimicrobial and antioxidant activity possessed by P. integrifolia may accelerate wound healing.

Objectives: To assess the wound healing activity of Premna integrifolia extract (PIE) by employing in-vivo experimental animal models and an in-vitro migration scratch assay. Furthermore, to assess its cytotoxicity using the MTT assay.

Methods: Wistar albino rats were used for the in vivo wound healing models. The animals were divided into four groups at random: Group I was untreated. Group II was vehicle control (ointment base). Group III was PIE ointment (5% W/W). Group IV was standard (povidone-iodine ointment) (5% W/W). The ointments were applied directly to the wounds as described above until they healed completely. The wound contraction percentage and tensile strength were calculated. The MTT test was used to determine the viability of the test extract against the fibroblast cells. The scratch assay was used in vitro to determine the wound healing potential of the test drug. P ≤ 0.05 values were considered statistically significant.

Results: Premna integrifolia extract did not possess any noticeable cytotoxicity to the cell line and showed an IC₅₀ of 185.98 μg/ml. The wound contraction potential of PIE ointment-treated animals was considerably greater (P ≤ 0.001) on days 4, 8, 12, 16, and 20 when compared to the control group. The percentage of wound contraction on day 20 was 99.92% in PIE-treated animals compared to 83.23% in untreated animals. Compared to the untreated group, the duration of full epithelization was significantly (P ≤ 0.01) shorter in the test group. When compared to the incision control group, the animals treated with PIE ointment had significantly higher (P ≤ 0.001) tensile strength. In addition, animals given the test drug had a significant (P ≤ 0.001) increase in total protein and hydroxyproline. In the in vitro scratch assay, test drug-treated cells demonstrated greater cell migration. Histology images confirmed that the test drug-treated group had epithelial tissue proliferation and keratinization.

Conclusion: The current study found that Premna integrifolia improved wound healing activity both in vitro and in vivo. These findings indicate that Premna integrifolia extract has wound-healing potential and could be a viable source of nutraceuticals with wound-healing properties.

Figure 1

(a) Circular excision wound on day 0. (b) Linear incision wound on day 0.

Figure 2

Pictographs. (a) Untreated. (b) Premna integrifolia extract (250 μg/ml). (c) Cisplatin.

Figure 3

Photos depicting the effect of PIE ointment on excision wound model across different stages of the study.

Figure 4

The percentage contraction of the wound area was employed as an evaluation criterion for in vivo wound-healing activity using an incision wound model. Values are expressed as mean ± SEM for 6 animals per group. ^∗P < 0.01. ^#P < 0.001 compared with controls (ANOVA followed by post hoc tests for multiple comparisons).

Figure 5

(a) The hydroxyproline content of various animal groups' granulation tissues. (b) Protein content in the granulation tissues of different animal groups.

Figure 6

The effect of PIE ointment on tensile strength in incision wound model in Wister rats.

Figure 7

Photos depicting the effect of PIE ointment on the incision wound model across different stages of the study. (a) Wound suturing on day 0. (b) Wound-healing on day 8. (c) Wound-healing on day 12. (d) Wound-healing on day 16. (e) Tensile strength of test drug-treated animal wound on day 20 (wound did not open after applying 1090 g of weight). (f) Tensile strength of control animal wound on day 20 (sutured wound opened after applying 768 g of weight).

Figure 8

Microscopical photos illustrating Premna integrifolia's ability to heal wounds in vitro. Images were taken at 0, 6, 12, and 24 hours after the mice fibroblast cells were cultured in the presence or absence of the test and standard drugs.

Figure 9

Antimicrobial activity of P. integrifolia against C. albicans: control itraconazole (a) and drug-treated (b). B. cereus: control ciprofloxacin (c) and drug-treated (d). E. coli: control ciprofloxacin (e) and drug-treated (f).

Figure 10

Histopathology analysis of newly healed tissue on day 20 post-treatment with PIE ointment. (a) Group I (excision control). (b) Group II (base treatment). (c) Group III (PIE ointment). (d) Group IV (Standard-Povidone-Iodine).