Evaluation of wound healing activity of ethanol extract of Annona reticulata L. leaf both in vitro and in diabetic mice model

Abstract

Background and aim: The leaves of AnnonareticulataLinn (牛心果niú x ınguǒ; Bullock’s heart), a member of Annonaceae family, have been used extensively in folk medicine; however, its wound healing potential is yet to be explored. Our aim was to investigate the wound healing ability of A. reticulataleaf extract in vitro and in streptozotocin induced diabetic mice model. Material and methods: We observed the plant extract induced proliferation and migration of primary human dermal fibroblast (HDF), human skin fibroblast cell line (GM00637) and human keratinocyte cell line (HACAT). The expression of transforming growth factor beta (TGF-β), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), alpha smooth muscle actin (α-SMA), matrix metalloproteinases (MMP-2, MMP-9), collagen-1, collagen-3, focal adhesion kinase (FAK) were evaluated by Western blot and gelatin zymography. Excisional diabetic wound model was used for in vivo wound healing assay. Furthermore, we processed wound tissue for histological and immunohistochemical study. Result: A. reticulata L. leaf extract stimulates proliferation and migration of HDF, skin fibroblast and keratinocyte significantly in a dose dependent manner; expression of TGF-β, CTGF, VEGF, α-SMA, MMP-2, MMP-9, collagen-1, collagen-3, FAK increased. Additionally, an enhanced expression of phospho-SMAD2, phospho-SMAD3 in the treated cells indicated the activation of TGF-β signal transduction pathway, similarly increased expression of phospho-AkT suggested activation of PI3/AkT pathway. Expression of CTGF and α-SMA was also increased significantly in wound tissue. Mass spectrometric analysis revealed that mainly two compounds to be present in the extract: quercetin and β-sitosterol. Conclusion: Collective data suggest that A.reticulata leaf extract may have a stimulatory effect in diabetic wound healing.

Keywords

• Annona reticulata L. leaf extract
• Human fibroblast
• Human keratinocyte
• Diabetic mice
• Wound healing