Eleutherine indica L. accelerates in vivo cutaneous wound healing by stimulating Smad-mediated collagen production

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Abstract

Ethnopharmacological relevance

Eleutherine indica L. has been used for healing of wound, painful and irregular menstruation, dysentery and lesions, and topically used as antiseptic and antimicrobial agent in folk medicine. In the present study, methanolic extracts of Eleutherine indica was subjected to scientific investigation for in-vivo cutaneous wound healing in wistar rat.

Materials and methods

In-vivo wound healing activity of Eleutherine indica was evaluated by using circular excision experimental models, followed by histopathological and western blot analysis. The healing potential was comparatively assessed with a reference gentamicin sulfate hydrogel (0.01% w/w). Wound contraction measurement, hydroxyproline estimation and western blot for COL3A1, bFGF, Smad-2, -3, -4, and -7 were performed.

Results

The methanolic extract of Eleutherine indica showed accelerated wound healing activity as evidenced by fast wound contraction rate and higher hydroxyproline content of granulation tissue. Western blot revealed the Smad-mediated collagen production promoting property of Eleutherine indica methanolic extract. Histopathological examinations also supported the experimental findings.

Conclusion

The study revealed that Eleutherine indica promotes wound healing by augmenting Smad-mediated collagen production in wound granulation tissue.

Introduction

Eleutherine indica L. is a tropical herb of family Iridaceae mainly cultivated in Southeast Asia including India and China. In India, this plant is distributed in the North-East region, where the tribal population used it as a folklore medicine for the treatment of dysentery, wound, and inflammation. The bulbs of Eleutherine species are used as folk medicine in many parts of the world and as cuisine in some Asian countries. Eleutherine bulbosa (Miller) is used in painful and irregular menstruation, and intestinal disorders (Lin et al., 2002), as a vermifuge, abortive and antifertility agent (Alves et al., 2003) and has been examined for wound healing property (Villegas et al., 1997). Red bulb of Eleutherine americana Merr. is used as traditional medicine against breast cancer by local people of Kalimantan (Indonesia) and for its immunostimulant, anti-inflammatory, anti-tumor, anti-bleeding properties and cardio-protection in coronary heart disorders (Song et al., 2009). The latter species also has antibacterial and antiquorum sensing activities to Staphylococcus aureus and Streptococcus pyogenes (Ifesan et al., 2009). Species of Eleutherine are reported to contain naphthoquinones, elecanicin, eleutherol, isoeleutherol-one, anthraquinones and glycosides (Alves et al., 2003, Qui et al., 2005, Xu et al., 2006, Paramapojn et al., 2008).

Wound healing is a tangled process that involves distinct overlapping phases of homeostasis, inflammation, proliferation, and tissue re-modeling (Jie et al., 2007). Complex healing cascade starts from the moment of injury and end with scar formation. Tissue injury induce blood coagulation process which resulted in the formation of insoluble fibrin matrix. Inflammatory cells (neutrophiles and macrophages) remove the non-functional cells and destroy the possible infections in the fibrin matrix (Kondo and Ishida, 2010). Proliferation phase initiated by macrophages, are actively progressed by fibroblast cells under control of the complex interplay of growth factors. Finally, in the remodeling phase, collagen is cross-linked with the already existing collagen and protein molecules, and contributed to the strength of the scar tissue.

Based on the traditional claims that species of Eleutherine have several therapeutic properties, including wound healing and antimicrobial (Villegas et al., 1997, Ifesan et al., 2009), it was decided to determine the wound healing activity of Eleutherine indica as lead to discover new wound healing agent. Eleutherine indica is considered as one of the most important folklore medicinal plant used for dysentery and wound healing by the tribal population of North-East India and to the best of our knowledge it is not properly explored scientifically so far. The present study was aimed to explore the wound healing potential of methanolic extract of Eleutherine indica and the possible mechanism to rationalize its traditional use.

Section snippets

Reagents and chemicals

Tris–HCl, phenazine methosulfate fluoride (PMSF), Bradford reagent, acrylamide and glycine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Carbopol 934, propylene glycol, gentamicin sulfate, l-hydroxyproline, p-dimethyl-amino-benzaldehyde were obtained from Himedia Pvt. Labs. (Mumbai, India). All primary/secondary antibodies used were procured from Santa Cruz (Santa Cruz, CA, USA). PVDF membrane and Western Max-HRP-Chromogenic detection kit were purchased from Amresco (Solon Ind.

Extraction of phytochemicals

The powdered bulb sample (100 g) contained 4.08 g in petroleum ether, 1.03 g in chloroform, 14.83 g in methanol and 7.26 g in aqueous. The preliminary phytochemical investigation revealed the presence of steroids, flavonoids, terpenes, tannins and alkaloids in the bulb extract.

Acute skin irritation and toxicity

Since the concentrations were safe without any symptoms of irritation and inflammation, high concentration of 2.5% (w/w) hydrogel was selected for in vivo wound healing study.

Wound contraction

The wound appeared clean and free of exudates

Discussion

Wound healing is a cascade of regulated bio-molecular events for regeneration and restoration of the tissue, involves different phases of inflammation, granulation, collagenization, collagen maturation and scar maturation. Since the age of Ayurveda, plant medicine has been practiced for wound healing and a number of scientific studies also revealed the potential of plant medicines for wound healing. Plant based medicines are largely preferred because of the perceived absence of unwanted

Conclusion

The present study revealed that the topical application of Eleutherine indica methanolic extract caused wound healing and increased the hydroxyproline content as compared to control groups. Histopathological findings also suggested the greater cell infiltration and collagen density with more regularly organized dermis. Eleutherine indica methanolic extract treatment also caused high expression of bFGF, COL3A1 and Smad-2, -3, -4 proteins with concurrent decrease in the expression of Smad-7.

Acknowledgment

Financial support to A.U. in the form of Research Fellowship by the DRDO, Ministry of Defence, Government of India is gratefully acknowledged.

References (19)

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