ฤทธิ์ส่งเสริมการหายของแผลของตำรับยาสมานแผลในหนูเบาหวาน ประเภทที่ 2 (Goto-Kakizaki rats)

Abstract

Diabetic foot ulcers (DFUS) has been noted as the most common cause of amputations occurring at least one in 10 people with diabetes. Despite the existence of standard wound care protocols, there is a rising interest in testing and exploring the possibility of medicinal plants and traditional medicines that can serve as effective natural wound healing products for the treatment of DFUS. This study focuses mainly on the wound healing mechanisms of Thai traditional medicine named Ya-Samarn-Phlae which has been prescribed by Mr. Somporn Chanwanisakul, a folk healer in the Southern Thailand. This present work aimed to (1) evaluate the pharmacognostic properties and phytochemical constituents of Ya-Samarn-Phlae's herbal ingredients which are Curcuma longa L. (Rhizome), Areca catechu L. (Seed), Oryza sativa L. (Seed), and Garcinia mangostana L. (Pericarp), (II) determine the contents of biological markers and wound- related in vitro biological activities of the ethanol extract (E-YaSP), infused oil which is the traditional form (T-YaSP), and ointment containing Ya-Samarn-Phlae (YaSP), (III) examine in vivo anti-inflammatory mechanisms of YaSP and T-YaSP, and (IV) describe wound healing mechanisms of YaSP and T-YaSP toward excision wound model in Wistar and non-obese type 2 diabetic Goto-Kakizaki (GK) rats. The pharmacognostic parameters of Curcuma longa, Areca catechu, Oryza sativa, and Garcinia mangostana were evaluated in accordance with either Thai herbal pharmacopeia or the Ayurvedic herbal pharmacopoeia. The amount of their impurities such as foreign matters, total ash content, acid insoluble ash, and percentage of loss on drying were found to be within the limit as per the specification in the pharmacopeias. Similarly, their solvent-soluble extractive values that use for estimating the amount of their active constituents were found to meet the required criteria. Liquid chromatography mass spectrometry profiling highlights that E-YaSP, T-YaSP, and YaSP contains at least 37, 30, and 19 the known constituents, respectively. Among these, alpha-mangostin and curcumin were selected as representative biological markers since these compounds has been found in all Ya-Samarn-Phlae preparations. The quantitative analysis using high performance liquid chromatography revealed that the contents of alpha-mangostin and curcumin in YaSP was found to be approximately 6 times lower than that of T-YaSP. Moreover, it should be noted that the contents of alpha-mangostin and curcumin in YaSP were more dramatically decreased than that of T-YaSP during the storage period of 6 months. In vitro biological assessments have demonstrated that E-YaSP, T-YaSP, and YaSP possessed nitric oxide free radical scavenging ability and lipid peroxidation inhibition, moreover, E-YaSP enhanced human dermal fibroblast migration a scratch wound closure assay and inhibited the nitric oxide production in RAW 264.7 cells stimulated by lipopolysaccharide. Both T-YaSP and YaSP were determined for skin irritation using albino rabbits and categorized based on Globally Harmonized System of Classification Toxicity as a category IV (non-to slightly irritating). In vivo anti-inflammatory mechanisms were described using a carrageenan-induced paw edema model in Wistar rats. Topical administration with YaSP and T-YaSP have shown significant (p< 0.05) inhibition of paw oedema on 1st hour after injected with carrageenan. These results were found in a similar pattern with paw edema inhibition was seen in phenylbutazone treatment. The levels of inflammatory and oxidative stress markers which are malondialdehyde (MDA), nitric oxide (NO), and myeloperoxidase (MPO) measured by enzyme-linked immunosorbent assay (ELISA) were significantly decreased in T-YaSP and YaSP treated groups and these levels were found to be comparable with phenylbutazone treated group. Furthermore, the results of this study have shown that both treatment with both T-YaSP and YaSP dramatically reduced the pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-alpha), Interleukin 1 beta (IL-1beta), and prostaglandin E2 (PGE2) and inflammatory enzymes including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in inflamed paw tissue. In conclusion, T-YaSP and YaSP may enhance the wound healing process via attenuating its inflammatory phase either by inhibition of nitric oxide production in the inflammatory mode of the macrophages or by causing reductions in the levels of inflammatory cytokines and enzymes. To further evaluated the wound healing property and described the mechanisms of action of Ya-Samarn-Phlae preparations, excision wounds were created in six groups consisting of 9-10 rats each. The first two groups received vehicles for YaSP and T-YaSP, while there were two groups received a commercially available traditional product, ThongnoppakhunR (as a positive control for T-YaSP) or Solcoseryl ointmentR (as a positive control for YaSP), and the last two groups received YaSP and T-YaSP topically every other day. The percentage of wound contraction on days 3, 5, 7, 9 and 11 as well as the levels of transforming growth factor beta-1 (TGF-beta1), vascular growth factor (VEGF), collagen type I, and collagen type III and histopathology parameters of healed wounds on 7th and 11th (for non-diabetic wounds) and on 11th (for diabetic wounds) were evaluated. The percentage of wound contraction and the reduction of wound area in the excision wounds were significantly different (p<0.05) between diabetic and non- diabetic rats, whereas notable wound healing effects of both YaSP and T-YaSP elucidated by the reduction of wound area were found to be higher than that of the vehicle controls. In addition, the changes in growth factors including TGF-beta1 and VEGF and the levels of collagen type I and III in YaSP and T-YaSP treated groups were comparable with the positive controls. According to our data, YaSP and T-YaSP could be noted as an effective natural wound healing preparation for both diabetic and non-diabetic wounds. This traditional medicine promotes skin wound healing, possibly by inhibiting the excessive and prolonged inflammatory phase and enhancing both proliferation and remodeling phases of acute wounds.