Purpose: An investigation was carried out to determine the effect of an ethanolic extract of the natural product of Daun sirih or Piper betle leaves on the effectiveness of plasma jet treatment for cutaneous acute wound healing in a small animal model mimicking a clinical setting. Method: An atmospheric plasma jet using medical grade argon gas as a carrier gas was developed. The ethanolic extract of Piper betle leaf (EPB) was formulated. Optical emission spectroscopy and chemical methods were applied to evaluate the presence of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the gas phase and in aqueous and ethanolic media. Small animals were classified into 5 groups, namely, Control (C), Plasma jet (P), Ethanolic extract of Piper betle leaf (EPB), Plasma jet followed by EPB (P-EPB) and EPB followed by plasma jet (EPB-P). The contact and meander styles of plasma jet treatment for wounds were applied daily on acute wounds for 1 min, either alone or before or after EPB treatments. Visual evaluation of wounds was conducted for 14 days. Microscopic evaluation was conducted on days 7, 11 and 14. General staining, namely, haematoxylin-eosin and Azan staining, was conducted to evaluate neoepithelialisation and new collagen formation. Results: This research showed that wound healing in the P group was faster than that in the other groups, while that in groups containing EPB was the same as that in C. In the P group, the number of days to reach peak inflammation was the fewest. On day 7, neoepithelialisation and new collagen formation in P were significantly higher than those in other groups. Conclusion: Plasma jet treatment alone is able to promote inflammation, neoepithelialisation and new collagen formation to accelerate acute wound healing; however, its admixture with EPB may impede such effectiveness. Based on the characterization of the ROS and RNS results, the ethanol solvent may play a primary role in impeding its effectiveness.
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