| Pine needles (PN) are leaves on pine trees belonging to the Pinaceae family. These natural plant resources haven’t been utilized fully for a long time depite their high nutritional and medicinal value. In terms of practical application, steam flash explosion (SFE) was employed to assist the extraction of flavonoids from pine (Larix olgensis Henry) needles, followed by the investigation of parameters optimization of extraction. Total flavonoids extract from pine needles was preparative purified by macroporous resin and its antioxidant activities and inhibitory effect on digestive enzymes were also investigated. The present study laid a foundation for PN-related product development.Firstly, SFE was employed to assist the extraction of flavonoids from PN, followed by the investigation of parameters optimization of extraction, and was compared with solvent extraction, microwave and ultrasound-assisted extraction. Scanning electron microscopy (SEM) and High performance liquid chromatography combined with photodiode-array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) were used to characterize the morphological changes and analyze flavonoids of PN before and after SFE treatment. The results indicated that after soaking for1.5h and SEF treated at1.5MPa for40s, followed by optimized extraction parameters, i.e. solid-liquid ratio1:40(w/v), ethanol concentration60%(v/v), temperature70℃, extraction time40min, extraction times once, total flavonoids extracted reached5.21±0.07%. SFE exhibited higher extraction yeild and efficiency compared with solvent extraction, microwave and ultrasound-assisted extraction. PN changed into an amorphous state and even particles after SEF treatment. The SEF-treated PN extract presented a much higher quantitative aglycone profile and lower glycosides profile in comparison with the untreated PN.Secondly, the adsorption capacity, desorption capacity and desorption ratio of seven kinds of resins for the flavonoids were evaluated. Adsorption and desorption kinetics on resins D101and DA201was investigated. Dynamic adsorption and desorption experiments were carried out on DA201resin packed column to obtain optimal parameters for purifying flavonoids extract from PN. Major flavonoids of purified flavonoids extract from PN were tentative identified by two-dimensional thin-layer chromatography and HPLC-DAD-ESI-MS. The results demenstrated that the optimum parameters for adsorption were as follows:the concentration of total flavonoids in sample solution3.27mg/mL, processing volume3BV, flow rate,1BV/h, and temperature25℃; for desorption, ethanol-water (50:50, v/v),4BV as an eluent, flow rate:4BV/h. After purification with DA201resin, the content of total flavonoids reached47.63±2.51%, with recovery yield of80.92±1.27%. Major flavonoids of purified flavonoids extract were apigenin-6-C-glucose, quercetin-3-O-glucose, kaempferol3-O-glucose and kaempferol.Finally, the antioxidant activities of purified flavonoids extract were analysed by1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and hydroxyl radical-scavenging assay, also its inhibitory effect on a-amylase and pancreatic lipase. The results showed that purified flavonoids extract exhibited strong antioxidant activity in scavenging DPPH and hydroxyl radical. The DPPH radical-scavenging ability of purified flavonoids extract was not as good as that of vitamin C, while for hydroxyl radical-scavenging ability9.05times as vitamin C. Purified flavonoids extract showed good inhibitory effect on pancreatic a-amylase and lipase. The inhibition of a-amylase and lipase by purified flavonoids extract was mixed inhibition. Purified flavonoids extract could significantly decrease the Michaelis-Menton constant and maximum reaction rate of the enzymatic reaction by its higher affinity for the enzyme-substrate complex. |
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