Von der Ethnopharmakologie zur rationalen Phytotherapie: Phytochemische Charakterisierung und präklinische Untersuchungen zur potenziellen Wundheilungsaktivität von ­Extrakten aus Combretum mucronatum Schum. & Thonn.

Zusammenfassung

Blattextrakte aus Combretum mucronatum Schum. & Thonn. werden in Westafrika ­traditionell zur Wundheilungsförderung verwendet. In der vorliegenden Untersuchung sollte die Droge phytochemisch charakterisiert, eine validierte HPLC-Methode zur Qualitätskontrolle der Droge entwickelt und funktionale Studien an humanen primären Hautzellen unter In-vitro-Bedingungen durchgeführt werden, um das Potenzial der Droge besser abschätzen zu können.

Ergebnisse: Detaillierte phytochemische Untersuchungen von wässrigen, hydroalko­holischen, Dichlormethan- und Petrolether-Ex­trakten der Blätter aus C. mucronatum ­ergaben die in Tabelle 1 aufgelisteten Verbindungen. Epicatechin, Procyanidin B2, ­Vitexin und Isovitexin wurden als relevante Leitsubstanzen ausgewählt, die gemäß ­einer nach ICH2-Guidelines validierten UHPLC-­Methode quantifiziert werden können. Die Analyse verschiedener Drogenchargen ­belegte höhere Gehalte der Leitsubstanzen in reifem Blattmaterial als in jungem. Die Trocknung der frisch geernteten Blätter bei erhöhter Temperatur führt zu Verlusten der Leitsubstanzen.

Während ein wässriger Extrakt (0,1-100 μg / ml) keinen Einfluss auf die Zellvitalität dermaler Fibroblasten und Keratinozyten zeigte, wurde die zelluläre Proliferation signifikant inhibiert (100 μg / ml). Der Extrakt ­stimulierte die terminale Differenzierung primärer Keratinozyten (1, 10 μg / ml). Dies bedeutet, dass der Extrakt den Zellzyklus stoppt, die Zellteilung unterbindet und die Zelle in die Differenzierung hin zu epithelialen Barrierezellen triggert, was als entscheidender Faktor für die finale Wundheilung bewertet wird. Im Rahmen ausgedehnter Struktur-Wirkungsbeziehungen wurde Procyanidin B2 als für diese Differenzierungsinduktion verantwortlicher Naturstoff aus dem Extrakt identifiziert.

Schlussfolgerung: Die In-vitro-Effekte des wässrigen Extraktes auf humane Hautzellen unterstützen die traditionelle Anwendung der Droge zur Wundheilung. Basierend auf den Daten zum potenziellen Wirkmechanismus erscheint der Einsatz von Zubereitungen aus C. mucronatum in dieser Indikation gerechtfertigt.

Summary

Ethnopharmacology as a basis of scientific phytotherapy: Phytochemical charac­terization and pre-clinical studies of the potential of wound healing activity of leaf extracts from Combretum mucronatum Schum. & Thonn.

Leaves from Combretum mucronatum Schum. & Thonn. are traditionally used for wound healing in Western Africa. Aqueous and hydroalcoholic extracts of the dried leaves have recently been shown to stimulate viability of human keratinocytes and dermal fibroblasts. The present study aimed to characterize the herbal material phytochemically, to develop a validated HPLC method for quality control of the herbal material, and to investigate the underlying pharmacological mechanism under in vitro conditions to understand the impact of C. mucronatum extracts on human skin cells.

Materials and methods: Extracts obtained from the leaves of C. mucronatum by ­using solvents with different polarity (petroleum benzine, dichloromethane, ethanol-water 50 %, water) were investigated concerning phytochemical composition by GC-MS, LC-MS and in part after fractionation and isolation of purified compounds. For quality control of the herbal material an ICH-2 validated UHPLC method was developed for quantification of the lead compounds epicatechin, procyanidin B2, vitexin and isovitexin. In vitro studies were performed using HaCaT keratinocyte cell line, primary keratinocytes and primary skin fibroblasts with determination of viability (MTT assay), cell proliferation (BrdU incorporation ELISA), cell toxicity (LDH release) and keratinocyte differentiation, using involucrin and keratin K10 as differentiation marker (confocal laser scanning microscopy, Western blot).

Results: A detailed phytochemical composition analysis of aqueous, hydroalcoholic, CH₂Cl₂ and petroleum benzine extracts from the leaves of C. mucronatum was performed and epicatechin, procyanidin B2, vitexin and isovitexin are assessed to be the lead compounds of the polar extract. Quantitative UHPLC investigations indicated mature leaves to have higher polyphenol content in comparison to young leaves. The drying process of the plant material was shown to have great influence on the content of the lead compounds. The aqueous extract (0.1 to 100 μg / mL) did not change cell viability of dermal fibroblasts and keratinocytes but inhibited cellular proliferation rates significantly at 100 μg / mL. The extract stimulated cellular differentiation of primary keratinocytes significantly at 1 and 10 μg / mL. Procyanidin B2 at 1 and 10 µM was shown to be responsible for the induction of this cellular differentiation, while epicatechin, and procyanidins B5, C1 and D1 were inactive.

Conclusion: The in vitro effects of the aqueous extract on the skin cells rationalized the remedial effect in wound healing and possibly accounts for the reason why this plant may be widely used for this purpose. On the basis of this study extracts from the leaves of C. mucronatum therefore have potential for the use in wound healing.

Schlüsselwörter

Combretum mucronatum - Combretaceae - Procyanidine - Keratinozyten - Differenzierung - Wundheilung

Keywords

Combretum mucronatum - Combretaceae - procyanidins - keratinocytes - differentiation - wound healing

¹ Universität Münster, Institut für Pharmazeutische Biologie und Phytochemie, Corrensstr. 48, 48149 Münster

² Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

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