Elsevier

Journal of Ethnopharmacology

Volume 134, Issue 3, 12 April 2011, Pages 1033-1038
Journal of Ethnopharmacology

Ethnopharmacological communication
In vitro and in vivo evaluation of the wound healing properties of Siegesbeckia pubescens

https://doi.org/10.1016/j.jep.2011.02.010Get rights and content

Abstract

Ethnopharmacological relevance

Siegesbeckia pubescens (SP) has been traditionally used as a wound healing agent.

Aim of the study

Investigate in vitro and in vivo healing properties of SP extract.

Materials and methods

The methanolic extract of SP was tested for the ability to stimulate the growth of mouse fibroblast NIH3T3 in vitro. The viability and proliferation of fibroblasts were evaluated at 72 h after cell seeding by MTT assay at 570 nm. To study wound healing properties in vivo, excision and incision wound models were used on rats and SP (3, 4, 5%, w/w) was topically administered. After treatment, wound contraction, epithelialization period and hexosamine content were evaluated in the excision wound model. In the incision wound model, wound sites were removed for histopathological analysis and skin-breaking strength determination.

Results

The methanol extract showed significant stimulation of the growth of mouse fibroblast NIH3T3 at 0.5–100 μg/mL. In excision wound, animals treated with 4, 5% (w/w) SP exhibited significant increases in the rate of wound contraction, period of epithelialization and content of hydroxyproline. In incision wound, the animals treated with both the 4 and 5% (w/w) SP extracts showed an increase in breaking strength when compared with the control, which was additionally supported by histopathological studies.

Conclusion

The experimental data revealed that the methanolic extract of SP displayed remarkable wound healing activity, corroborating its traditional use.

Introduction

Since ancient times, people have used plants and preparations thereof to accelerate the wound healing process (Fronza et al., 2009, Reuter et al., 2009). Often their use is merely based on tradition, without any scientific evidence of efficacy and little knowledge about putative active compounds or their mode of actions.

Siegesbeckia pubescens (SP) originates in South-East Asia, and grows well in particularly hot, damp climates. This herb is an important traditional Chinese medicine widely used in the treatment of rheumatic arthritis, hypertension, malaria, neurasthenia and snakebite (Zheng, 2005). Its aerial part has also been used for centuries externally to stimulate wound healing (Park et al., 2007). In spite of its wide use over a long period of time, no systematic approach has been made to study the wound healing activity of this plant.

Wound healing consists of an orderly progression of events that re-establish the integrity of the damaged tissue: inflammatory, proliferation and remodeling stages (Kokane et al., 2009). The inflammation stage begins immediately after injury, first with vasoconstriction that favors homeostasis and releases inflammation mediators. The proliferative phase is characterized by granulation tissue proliferation formed mainly by fibroblast and the angiogenesis process. The remodeling stage is characterized by reformulations and improvement in the components of the collagen fibre that increases the tensile strength (Varoglu et al., 2010).

Thus, it is generally believed that alterations in the first phase, which is the inflammatory one, will impact the overall integrity of the healing wound (Schultz et al., 2003, Süntar et al., 2010). In our previous study, SP was investigated for antiinflammatory activity. The results confirmed that the use of SP can inhibit the topical inflammation (Wang et al., 2008). In this study, our objective was to investigate the stimulation of growth of fibroblasts as an aspect of promotion of wound healing in vitro and to evaluate the wound healing activity of SP in vivo on various would healing phases.

Section snippets

Plant material

The aerial part of SP used in this study was collected from its natural habitat in Hubei province, China, in autumn 2009, and as authenticated by Professor Jia-Chun Chen (School of Pharmacy, Huazhong University of Science and Technology, China). A voucher specimen (No. 091011-S) was preserved at the herbarium of School of Pharmacy, Huazhong University of Science and Technology. The plant material was air dried in shade, powdered by a mechanical grinder, passed through a 40-mesh sieve and stored

MTT assay

The growth of fibroblasts was observed by visual examination through inverted phase contrast microscope at a magnification of 400× at 48 h. Fibroblast cells NIH3T3 were spindle shaped, elongated and bipolar in nature.

MTT assay results are shown in Fig. 1 and are given as sample absorbance/control absorbance versus concentrations. Those with sample absorbance/control absorbance values higher than 1 were accepted to have growth stimulant activity. The extract showed significant stimulation of the

Discussion

Experiments, extended on cell cultures are currently one of the most popular and effective methods to test the sensitivity of selected group of cells to substances present in their microenvironment. Fibroblast cell cultures have been proposed as a method for testing wound-healing activity in vitro (Abe et al., 2000). We firstly developed fibroblast culture technique to evaluate the SP extract.

In our in vitro experiments we used MTT test. This cell proliferation assay was performed after seeding

Conclusion

From these overall results, we can conclude that the methanolic extract of Siegesbeckia pubescens showed stimulation of the growth of mouse fibroblasts at 0.1–100 μg/mL in vitro, the topical application of at least 4–5% methanolic extract of Siegesbeckia pubescens produced significant wound healing activity. The results reported here corroborate the potential of Siegesbeckia pubescens extract as an agent for wound healing. This could mean that the methanol extract contains high concentration of

Acknowledgements

Financial supports from the Important National Science and Technology Specific Projects (No. 2009ZX09301-014) and the Science Research Foundation of Health Department of Hubei Province (No. 2008Z-Y20) are gratefully acknowledged.

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