Ellagitannins from Phyllanthus muellerianus: geraniin stimulates keratinocytes differentiation and collagen synthesis of skin dermal fibroblasts - new concept for improved wound-healing

Agyare C, Deters A, Lechtenberg M, Petereit F, Hensel A

Zusammenfassung

Leaves from Phyllanthus muellerianus (Kuntze) Exell. are traditionally used for wound healing in Western Africa. Aqueous extracts of dried leaves recently have been shown to stimulate proliferation of human keratinocytes and dermal fibroblasts. Within bioassay-guided fractionation the ellagitannins geraniin (1), corilagin (2), furosin (3), the flavonoids quercetin-3-O-β-d-glucoside (isoquercitrin), kaempferol-3-O-β-d-glucoside (astragalin), quercetin-3-O-d-rutinoside (rutin), gallic acid, methyl gallate, caffeic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeoylmalic acid (phaselic acid) have been identified in P. muellerianus for the first time. Geraniin was shown to be the dominant component of an aqueous extract. Suitable analytical methods for quality control of geraniin in P. muellerianus extract (methanol/water, 70/30) have been developed and validated based on ICH guidelines (ICH-compliant protocol). Geraniin and furosin increased the cellular energy status of human skin cells (dermal fibroblasts NHDF, HaCaT keratinocytes), triggering the cells towards higher proliferation rates, with fibroblasts being more sensitive than keratinocytes. Highest stimulation of NHDF by geraniin was found at 5μM, and of keratinocytes at 50-100μM. Furosin stimulated NHDF at about 50μM, keratinocytes at about 150-200μM. Necrotic cytotoxicity of geraniin, as measured by LDH release, was observed at 20μM for NHDF and 150μM for keratinocytes. Toxicity of furosin - less than that of geraniin - was observed at >400μM. Furosin and geraniin stimulated the biosynthesis of collagen from NHDF at 50μM and 5-10μM respectively. Geraniin at 105μM significantly stimulated the differentiation in NHEK while furosin had a minor influence on the expression of involucrin and cytokeratins K1 and K10. The study proves clearly that hydrophilic extracts from P. muellerianus and especially the lead compound geraniin exhibit stimulating activity on dermal fibroblasts and keratinocytes, leading to increased cell proliferation, barrier formation and formation of extracellular matrix proteins. From these findings the traditional clinical use of such extracts for wound healing seems to be justified.