Enzymatic hot pressurized fluids extraction of polyphenolics from Pinus taiwanensis and Pinus morrisonicola

This study was engaged in application of enzymatic hydrolysis on the hot pressurized fluid extraction (HPFE) to isolate the polyphenols from the needles of Pinus taiwanensis (PT) and Pinus morrisonicola (PM). This HPFE process was investigated using an L18 orthogonal experimental design. Our experimental results indicate that a mixed enzyme of cellulase, hemicellulase, pectinase and protease is able to enhance cell breakage during the sample pretreatment and the HPFE process. A suitable dynamic enzymatic HPFE conditions were 1.5% wt of mixed enzymes in 70% ethanol (pH 6) at a flow rate of 10 mL/min, under pressure 689 kPa and 343 K for 180 min. The content of polyphenols obtained from the dynamic mixed enzymatic hydrolysis HPFE process is higher than that obtained from the static enzymatic hydrolysis HPFE process at 4137 kPa and 343 K for 180 min. Anti-oxidative activities of the ethyl acetate fraction from partition of the resultant of the dynamic enzymatic HPFE process determined by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, peroxide radical release inhibition, nitrite oxide radical scavenging and photochemiluminescence were highly correlated with the content of polyphenols in the extracts. The extracts of PT needles have higher chemical antioxidant activities than that obtained from PM needles. However, in terms of antioxidant abilities from water-soluble and lipid-soluble fractions determined by photochemiluminescence method, the PM needles extracts demonstrated better activity than that from the PT needles extract. In the bioassay tests, the extracts of PM and PT needles demonstrate only a little viability of normal control cells at doses of 8.59 μg/mL and 5.86 μg/mL, respectively, but exhibit 10% and 16% of growth inhibition effect on DBTRG-glioblastoma cell of the human brain.