Separation of nine novel triterpene saponins from Camellia japonica seeds using high‐performance countercurrent chromatography and reversed‐phase high‐performance liquid chromatography.

Introduction: Camellia japonica L. (Theaceae) is an evergreen shrub, which is cultivated as a popular ornamental tree in Korea, China, and Japan and its seeds have been used as a source of cooking oil, in cosmetics and as a traditional medicine. Intensive phytochemical works have revealed that oleanane‐type saponins are the characteristic compounds of the seeds of C. japonica. Objective: The purpose of the present study is to isolate and determine oleanane‐type saponins from C. japonica using high‐performance countercurrent chromatography (HPCCC) coupled with reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and spectroscopic evidences, respectively. Methodology: HPLC electrospray ionisation quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF‐MS) was applied to profile the saponin composition of an enriched saponin extract of C. japonica seeds. The enriched saponin extract was separated by HPCCC using a dichloromethane/methanol/isopropanol/water (9:6:1:4, v/v/v/v) system and RP‐HPLC. The structures of the isolates were determined utilising ESI‐Q‐TOF‐MS, one‐dimensional and two‐dimensional NMR and optical rotation. Results: HPCCC on enriched saponin extract of C. japonica yielded four saponin fractions in the order of the number of sugars attached to the triterpene aglycone, and preparative RP‐HPLC on each saponin fraction led to the isolation of nine novel saponins, namely camoreoside A–I, along with six known ones. Conclusions: This study indicates that combination of HPLC‐ESI‐Q‐TOF‐MS analysis and HPCCC coupled with RP‐HPLC are excellent tools for discovering saponins from natural sources. High‐performance liquid chromatography electrospray ionisation quadrupole time‐of‐flight mass spectrometry (HPLC‐ESI‐Q‐TOF‐MS) revealed that enriched saponin extract of Camellia japonica contained a variety of saponins with di‐, tri‐, tetra‐saccharides as oligo‐glycoside moieties. High‐performance countercurrent chromatography (HPCCC) coupled with reversed‐phase (RP) HPLC were successfully applied to isolate nine new saponins namely camoreoside A–I along with six known ones from enriched saponin extract of C. japonica seeds. The structures of the isolates were determined utilising ESI‐Q‐TOF‐MS, one‐dimensional and two‐dimensional NMR and optical rotation. [ABSTRACT FROM AUTHOR]