Study On The Chemical Compositions And Biological Activity From Pine Needles Of Pinus Massoniana L.

In this thesis, the protein tyrosine phosphatase 1B(PTP1B) inhibitory activity ingredients in the pine needles of Pinus massoniana L.(PNPM) were studied systematically. The research includes two parts. First, the chemical compositions were analysed and predicted for PTP1 B inhibitory activity in the essential oil of PNPM. Second, the active compound in the ethyl acetate extracts of PNPM was separated through high-speed countercurrent chromatography(HSCCC).Firstly, a new method based on GC-MS coupled with molecular docking was established for analysis, identification and prediction of the PTP1 B inhibitors in the essential oil from PNPM. The essential oil from PNPM with the value of 25.46±0.01 μg/mL exhibited the potential inhibitive activity against PTP1 B in vitro. After GC-MS analysis, thirty-six compounds were identified from this oil according to references, retention indices and mass spectra data. The results indicated that the major compounds in the essential oil were terpenoids, such as β-pinene(39.24%), α-pinene(14.68%) and germacrene(9.08%). The identified compounds were docked with the target of PTP1 B by the means of molecular docking. Manoyl oxide with the lowest binding energy of-7.02 kcal/mol were wrapped completely by the active site of PTP1 B. The docking results indicated that manoyl oxide had the potential PTP1 B inhibitory activity and may be responsible for the PTP1 B inhibitory activity of the essential oil from PNPM.Secondly, a HSCCC method combined with the consecutive injection and the two-phase solvent system preparation using UNIFAC mathematical model was successfully developed for separation and purification of tiliroside from ethyl acetate extracts of PNPM. HSCCC was performed with a two-phase solvent system composed of n-hexane-ethyl acetate-methanolwater(3:5:3:5, v/v) at a flow rate of 6 mL/min. The components of the two-phase solvent system were calculated according to UNIFAC mathematical model to prepare the stationary and mobile phases, separately. Only 300 mL stationary phase was filled into the column, and the crude sample(200 mg, ten times) was consecutive loaded into the column at the given interval time and eluted using the individually prepared mobile phase without reestablishing hydrodynamic equilibrium. After recrystallized, 168 mg tiliroside with the purity of 95.8% was obtained from 2 g ethyl acetate extracts of PNPM through the consecutive injection. The structure was identified by 1H NMR and 13 C NMR. The molecular docking result showed that tiliroside with the binding energy of-6.26 kcal/mol possessed the potential PTP1 B inhibitory activity, which was forthur verificated by in vitro PTP1 B activity assay. Tiliroside with the IC50 value of 12.04±0.09 μmol/L possessed the potential inhibitive activity against PTP1 B.