Exploring Traditional Japanese Herbal Ointments for Wound Healing: A Wide Diversity of Practices, Metabolites, and Effects.

Ethnopharmacological relevance: In Kampo Japanese traditional medicine, ointments are used to treat skin wounds and promote wound healing. These ointments are prepared by extracting herbal crude drugs in sesame oil, a technique that has been practised for centuries. Their preparation varies significantly in terms of ingredients, plant species, temperature, and extraction time, leading to a wide diversity of formulations. Unlike hydrophilic plant extracts, the phytochemical composition and wound-healing efficacy of sesame oil-based herbal extractions remain largely unexplored.

Objective: The objective of this study is to address this gap by focusing on lipophilic plant metabolites present in these topical formulations and to explore their potential contribution to wound healing using a keratinocyte model in vitro.

Materials and methods: A survey was conducted to document Kampo ointment-related practices, focusing on highlighting variations in extraction protocols, crude drug origins, and manufacturing practices. Sample preparation excluded solid compounds from the formulations and used different solvents for the metabolomic and biological aspects. Metabolomics profiling was performed using LC-HRMS and LC-HRMS/MS to analyse the impact of different preparation temperatures and botanical sources on extract composition. In vitro assays assessed keratinocyte migration and proliferation using scratch assays and EdU (5-ethynyl-2'-deoxyuridine) incorporation and data were evaluated with appropriate statistical methods.

Results: A diversity of practices concerning the Kampo ointments Shiunkō, and Shinsen taitsukō was identified. This mainly concerned the extraction temperature with three protocols: maceration (M), Low Heating (LH) and High Heating (HH). Significant differences in metabolite profiles were observed, particularly with regard to the heating temperature of the sesame oil and the origin of the crude drugs. These parameters markedly influenced the chemical composition through both the formation of transformed lipids and the degradation of bioactive compounds such as tocopherols and sterols. While the variety of sesame oils and Angelica acutiloba extracts showed no distinct biological effects on keratinocyte migration, the complete formulas Shiunkō and Shinsen taitsukō unexpectedly delayed wound closure in the model. This result is possibly due to the degradation of wound-healing metabolites and the presence of cytotoxic ones, or the inadequacy of the cell model used to evaluate these complex lipophilic preparations.

Conclusion: This study highlights the diversity and complexity of traditional Kampo ointments in terms of chemical composition and, to a lesser extent, biological activity. Preparation temperature and ingredient origin markedly shaped the metabolite profiles, affecting both the formation of new lipophilic compounds and the degradation of healing-related metabolites. Higher heating temperatures and longer extraction times promoted the formation of modified lipid compounds and the loss of bioactive metabolites, such as tocopherols and sterols. Meanwhile, the botanical origin of Angelica acutiloba affected the quantity of glycosylated terpenoids associated with wound healing. Complete formulas were found to reduce keratinocyte migration in an in vitro monolayer model. Targeted metabolomics suggested a loss of vitamin E-like hydroquinone lipids in Shiunkō and the presence of an esterified diterpene in Shinsen taitsukō as possible contributors. The direct clinical relevance of these observations remains uncertain. These results emphasise the need for in-depth investigations into crude drug oily extractions, which are still underexplored. Research into traditional Kampo ointments could benefit from a more integrated approach that combines metabolomics with innovative biological models, in order to better elucidate their therapeutic applications in wound care.