Research On The Antioxidant Constituents Of Pyrola Incarnata Fisch.in The Northeast Frigid Region Of China And Extraction Process Optimization

In the present study,the object of study was Pyrola incarnata Fisch.in the northeast frigid region of China.An efficient,fast and accurate method was developed for the analysis of active constituents in Pyrola incarnata Fisch..Through we studied of variation of active constituents and antioxidant activity in Pyrola incarnata Fisch.from different sites and different growing seasons in the northeast frigid region of China,the best harvest site and time of Pyrola incarnata Fisch.was comfirmed.The extraction process conditions of new green solvent-deep eutectic solvent based on microwave assisted extraction were optimized.A kind of cheap,strong adsorption ability,green adsorption material-magnetic cellulose beads was prepared.And used for enrichment and separation of main active components in Pyrola incarnata Fisch..The research results were as followed:1.A HPLC method was developed for the simultaneous analysis and determination of eight active constituents in Pyrola incarnata Fisch.,the chromatographic conditions as follows:Chromatographic column:KYA HIQ Sil C18W reversed-phase column(250 mm × 4.6 mm i.d.,5 ?m),flow rate 1.0 mL/min,column temperature 30?,injection volume 10?L,determine wavelength 254 nm,265 nm and 280 nm.Mobile phase consisted of 0.1%formic acid aqueous solution(A)and methanol(B),gradient elution was performed as follows:0-5 min,12-18%B;5-15 min,18-27%B;15-20 min,27%B;20-32 min,27-38%B;32-40 min,38-41%B;40-55 min,41%B;55-60 min,41-45%B;60-65 min,45-55%B;65-70 min,55%B;70-75 min,55-65%B;75-80 min,65-75%B;80-85 min,75%B.The HPLC were using for separation,identification and quantification of eight active constituents in Pyrola incarnata Fisch.for the first time.The developed method was high precision,good repeatability and high accuracy,which was suitable for the determination of active constituents in Pyrola incarnata Fisch..2.Variation in contents of active constituents and antioxidant activity in Pyrola incarnata Fisch.from different sites in northeast frigid region of China were investigated(1)The quality of Pyrola incarnata Fisch.from eight main production areas(Tahe,Xinlin,Tulihe,Jiagedaqi,Shengshan,Dayangshu,Wudalianchi and Wuying)in northeast frigid region of China were investigated.The samples from Tahe and Tulihe had the higher total phenolics and flavonoids contents.And these two regions had the better DPPH and ABTS radical-scavenging activity,The IC50 of Tahe samples determined by DPPH radical-scavenging activity was very close to that of positive control Vc.In addition,the samples of Tahe,Tulihe,Dayangshu and Xinlin possessed good reducing power,their capacities of reducing power were obviously better than those of positive control BHT.(2)HPLC analysis found that hyperin and 2'-O-galloylhyperin were the main active components in Pyrola incarnata Fisch.,and their contents obviously higher than the other active constituents.Principal component analysis(PCA)was performed to understand the interrelationships between antioxidant activities and active constituents.The results indicated that Pyrola incarnata Fisch.from Tahe had the best quality,and Tahe was developed as a good producing area for Pyrola incarnata Fisch..3.Variation in contents of active constituents and antioxidant activity in Pyrola incarnata Fisch.from different growing seasons in Northeast China were investigated(1)With the best quality in Tahe as the harvest site,the variations of active constituents and antioxidant activities of Pyrola incarnata Fisch.samples from May to October were determined.July,August and September possessed a higher total phenolic contents.The harvest samples of August and September had the excellent DPPH and ABTS radical scavenging capacity,reducing power and FRAP activity.The samples harvest from August and September showed obviously higher ABTS radical scavenging capacity and FRAP activity which was also higher than the positive control Vc.(2)The main active constituents of Pyrola incarnata Fisch.samples were analyzed by HPLC.Hyperin and 2'-O-galloylhyperin are main compounds of Pyrola incarnata Fisch..The contents of hyperin and 2'-O-galloylhyperin were higher in July,August and September samples.Evaluation of the quality with the samples from May to October using PCA,it indicated that the quality of Pyrola incarnata Fisch.samples collecting in August and September had better than those in other months.August and September were the suitable time for harvesting the Pyrola incarnata Fisch.samples.4.Deep eutectic solvent based on microwave assisted extraction of active compounds from Pyrola incarnata Fisch.were optimizedThe effects of the composition and proportion of polyol-deep eutectic solvent,adding water volume percentage,extraction temperature,extraction time and liquid/solid ratio on the extraction yield were studied.The optimum parameters were as follows:polyol-deep eutectic solvent ChCl/1,4-Butanediol ratio of 1/4(mol/mol),adding water volume percentage 30%,extraction temperature 70?,extraction time 20 min,liquid/solid ratio 10:1 mL/g.Under the optimal parameters,the average extraction yields of hyperin,2'-O-galloylhyperin,quercitrin,quercetin-O-rhamnoside and chimaphilin were 1.617,4.908,0.090,0.041 and 0.347 mg/g,respectively.5.Preparation of the new green efficient adsorption materials-magnetic cellulose beads.Preparation of cellulose beads with cotton as matrix,made by dissolving,viscose,reverse suspension polymerization process eventually formed cellulose beads.After that,the cellulose beads were removal of pore-foaming agent with diluted hydrochloric acid,modified by SOCl2 and then modified by a series of graft modification group,formed with different amine,amide and ester groups based grafting modification of cellulose beads.Magnetite Fe3O4 particles were prepared by chemical co-precipitating method on cellulose beads.The magnetic cellulose beads were characterized by FTIR spectroscopy,X-ray diffraction,scanning electron microscopy.All this experiments showed that graft modification group and Fe3O4 were successfully immobilized on the cellulose beads.At last,the results of VSM indicated that the preparation of immobilized Fe3O4 cellulose beads had super paramagnetic magnetic.Magnetic intensity:CB+Fe3O4 7.1197 emu/g?CB-amine group+Fe3O4 5.2476 emu/g?CB-amide group+Fe3O4 1.5765 emu/g and CB-ester group+Fe3O4 1.5097 emu/g.6.Application of magnetic cellulose beads completed enrichment and separation of the main active constituents in Pyrola incarnata Fisch.Comparsion of the adsorption and desorption capacity of ten common macroporous resins and seven magnetic cellulose beads on enrichment of hyperin and 2'-O-galloylhyperin.The results showed that adsorption capacities of magnetic cellulose beads were far higher than that of macroporous resins for two target compounds,and the adsorption capacity of CB-TETA+Fe3O4 was the largest.Research of adsorption kinetics and isothermal adsorption curve of CB-TETA+Fe3O4 indicated that the adsorption process can be better described by the second-order kinetics model than the first-order kinetics model.And the adsorption behavior is easy to occur.It is suitable for used as adsorbent adsorption of hyperin and 2'-O-galloylhyperin in Pyrola incarnata Fisch..In this study,it was completed that system evaluation of the quality of Pyrola incarnata Fisch.in the northeast frigid region of China and optimization of the extraction process of active components,the development and utilization of Pyrola incarnata Fisch.resources provides the necessary scientific basis,and also provides technical support for large-scale extraction,enrichment and separation of active ingredients of Pyrola incarnata Fisch..A new kind of adsorption materials-magnetic cellulose beads was prepared for the first time,it will provide a kind of new environmental adsorption material for enrichment and separation of natural products,its industrialization prospect is extremely broad.