Ixora coccinea Enhances Cutaneous Wound Healing by Upregulating the Expression of Collagen and Basic Fibroblast Growth Factor

Abstract

Background. Ixora coccinea L. (Rubiaceae) has been documented for traditional use in hypertension, menstrual irregularities, sprain, chronic ulcer, and skin diseases. In the present study, I. coccinea was subjected to in vitro and in vivo wound healing investigation. Methods. Petroleum ether, chloroform, methanol, and water sequential I. coccinea leaves extracts were evaluated for in vitro antioxidant, antimicrobial, and fibroblast proliferation activities. The promising I. coccinea methanol extract (IxME) was screened for in vivo wound healing activity in Wistar rat using circular excision model. Wound contraction measurement, hydroxyproline quantification, and western blot for collagen type III (COL3A1), basic fibroblast growth factor (bFGF), and Smad-2, -3, -4, and -7 was performed with 7-day postoperative wound granulation tissue. Gentamicin sulfate (0.01% w/w) hydrogel was used as reference standard. Results. IxME showed the potent antimicrobial, antioxidant activities, with significant fibroblast proliferation inducing activity, as compared to all other extracts. In vivo study confirmed the wound healing accelerating potential of IxME, as evidenced by faster wound contraction, higher hydroxyproline content, and improved histopathology of granulation tissue. Western blot analysis revealed that the topical application of I. coccinea methanol extract stimulates the fibroblast growth factor and Smad mediated collagen production in wound tissue.

Figure 1

Ferric ion reducing antioxidant power (FRAP) of different I. coccinea extracts.

Figure 2

(a) Effect of I. coccinea extracts on human dermal fibroblast proliferation. (b) Protection of human dermal fibroblast cells against H₂O₂-induced damage with simultaneous application of different I. coccinea extracts. FBS: Fetal Bovine Serum; Cat-Catalase. Values are expressed as mean ± SD. Asterisk (∗) indicates significantly different (P < 0.05) as compared to H₂O₂ treated negative treated groups.

Figure 3

Effect of I. coccinea methanol extract (IxME) on wound healing. (a) Pictorial representation of wound closure in wistar rat. (b) Wound contraction rate. (c) Hydroxyproline content. Values are expressed as mean ± SD. Asterisk (∗) indicates significantly different (P < 0.05) as compared to the nontreated and vehicle treated groups.

Figure 4

Microscopic view of healing wound granulation tissue and remodeling epidermis/dermis in (1) nontreated, (2) vehicle control, (3) IxME, and (4) gentamicin sulfate treated animal groups. Section shows the hematoxylin and eosin stained epidermis and dermis in (A) and (C) (100x) and Masson's trichrome stained dermis in (B) and (D) (400x) of 7- and 15-day postoperative treated animal groups, respectively. The arrows point to the events of wound healing—S: scab; U: ulcer; Re: reepithelization; F: fibroblast; PMC: polymorphonuclear cells; MNC: mononuclear cells; C: collagen; and NV: neovascularization.

Figure 5

Effect of I. coccinea methanol extract (IxME) on COL3A1 and bFGF Smad-2, -3, -4, and -7 protein expressions on day 7 (seven) in wound tissues, detected by western blot. Lane (1) nontreated, (2) vehicle control, (3) IxME, and (4) gentamicin sulfate treated animal group, respectively. Values are expressed as mean ± SD. Asterisk (∗) indicates significantly different (P < 0.05) as compared to the nontreated and vehicle treated groups. Hash (#) indicates significantly different (P < 0.05) as compared to gentamicin sulfate treated group.