Novel compounds discovery approach based on UPLC-QTOF-MS/MS chemical profile reveals birch bark extract anti-inflammatory,-oxidative, and-proliferative effects

Ethnopharmacological relevance: Betula pendula subsp. Mandshurica (Regel) Ashburner & McAll. Cortex (birch bark) is a globally traditional medicine for treating multiple inflammatory diseases. Its records are included in the Compendium of Materia Medica and other ancient medical literatures. However, uncovering its chemical profile and exploring novel biologically active compounds from birch bark remains a significant challenge.Aim of the study: To uncover the anti-inflammatory,-oxidative, and -proliferative mechanisms and potentially effective compounds of birch bark extract by combing chemical profiling, isolation, identification, together with in vivo, in vitro, and silico evaluation. Materials and methods: Ultra-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) was used to obtain the chemical profile of birch bark extract. The new compounds were obtained via column chromatography and analyzed using X-ray diffraction and electronic circular dichroism for absolute configuration confirmation. The zebrafish caudal fin inflammation-induced model, qPCR, and Western blot analysis were used to explore the effects and underlying mechanisms of birch bark extract. In vitro cytotoxicity assays and kinases screening conducted to gain preliminary insight into the anti-proliferative effects of birch bark extract and its isolated compounds. In addition, in-silico molecular docking was performed to investigate the putative mechanism.Results: UPLC-QTOF-MS/MS chemical profiles revealed 105 compounds in birch bark extract, with 80 of these were first reported in B. pendula subsp. Mandshurica cortex. We selected five compounds speculated as novel and isolated three ones (one triterpenoid derivative and two lupine series triterpenoids) for further analysis. Birch bark extract exerted antioxidative and anti-inflammatory effects on zebrafish, as shown by the downregulated reactive oxygen species levels and COX-2 alpha, IL-1 beta, and TNF-alpha expression, which occurred through NF-kappa B signaling pathway activation. The in vitro anti-proliferative effects of birch bark extract and compound 44 were also un-veiled. Moreover, the putative anti-tumor mechanism of compound 44 was revealed using kinase screening and in-silico molecular docking.Conclusions: This study provided a predictable chemical profile and demonstrated the pharmacological effects of birch bark extract, elucidated the mechanism of this traditional Chinese medicine and suggested it as a novel anti-cancer candidate.