| Rubus parvifolius L., a deciduous thorny shrub, is traditionally used as herbal medicine for the treatment of rheum, fever, angina, enteritis, hepatitis, concretion, dermatitis, and so on. Due to its various pharmacological activities, the characterization of bioactive constituents responsible for pharmacological effects of the traditional herbal drug is of great importance. High-performance liquid chromatography/tandem mass spectrometry (HPLC-MS"), as an efficient separation and analytical system, combining the high separation capability of HPLC and the power of MS in structural determination, has been widely applied in various fields such as natural product and drug analysis. HPLC-MSn was utilized to identify bioactive constituents from Rubus parvifolius L. extract and distinguish these isomeric ursane-type triterpenoids rapidly. The details are listed as follows:Part I The hepatoprotective and antioxidant activity of the n-butanol extract of Rubus parvifolius L. was evaluated. In this study, we evaluated the antioxidant activity of the extract by DPPH free radical scavenging assay and assessed its hepatoprotective effects on chronic CCl4-induced liver damage in mice by biochemical assays and histopathological observations. Results demonstrated that Rubus parvifolius L. extract possessed pronounced hepatoprotective effects against CCl4-induced hepatic injury, which was at least partially attributed to its strong antioxidant capacity. Phytochemical analysis based on HPLC-MS" revealed the presence of various phenolic compounds, including caffeic acid conjugates, ellagic acid glycosides, and flavonol glycosides, which might be responsible for the hepatoprotective and antioxidant activity of the extract. Furthermore, offline Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was also performed to assign accurate elemental compositions and confirm the proposed structures further.Part II A facile method was established for the differentiation of diastereoisomeric ursane-type triterpenoids from the EtOAc extract of Rubus parvifolius L.. Based on reference standards, the abundance of the fragment ion [M-H-H2O-HCOOH]- for 3β-OH compounds in the MS3 experiment was dramatically compared to [M-H-H2O-CO2]-, whereas for 3a-OH compounds, the product ion [M-H-H2O-CO2]- was noted to be higher than [M-H-H2O-HCOOH]-. Energy-resolved mass spectrometric experiments were carried out to support the differentiation of these diastereoisomeric triterpenoids at C-3 position. Using this method, a total of nine ursane-type triterpenoids from the EtOAc extract of Rubus parvifolius L., including four pairs of epimers at C-3 position, were identified and distinguished rapidly.
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