| Pinus koraiensis as one of the economical trees is widely distributed in the areas such as northeast China,Russia,North Korea,and so on.However,pinecones of P.koraiensis as the by-products in pine-nut processing were often used as fertilizer or even as garbage,leading to environmental pollution and a waste of resources.The comprehensive utilization of pinecones of P.koraiensis has not been resolved for a long time.Researches have confirmed that the extracts of P.koraiensis pinecones contained a lot of polyphenols including procyanidins.In this thesis,procyanidins from pinecones of P.koraiensis were extracted effectively by enzyme hydrolysis pretreatment ionic liquid-lithium salt based microwave-assisted extraction.Single factor experiment,Plackett-Burman design(PBD)and Box-Behnken design(BBD)were employed to optimize the experimental factors.Then high speed counter-current chromatorgraphy(HSCCC)was applied to separate procyanidins from crude extracts,and the most active fraction procyanidins F3 was selected with antitumor activity as the index.Gold nanoparticles coated liposomes were constructed as drug delivery system with procyanidins F3 as experimental subject.In order to promote the exploitation of natural products,red blood cell mimetic microcapsule was constructed based on layer-by-layer assembly method.The main contents of the thesis are as follows:Procyanidins were extracted by aqueous enzymatic pretreatment ionic liquid-lithium salt based microwave-assisted extraction method.Cellulase and pectinase(1:1,w/w),[C4min][Br] and Li Cl was selected as the optimum using single factor experiment.PBD and BBD were employed to screen the other factors including liquid-solid ratio,the amount of enzymes,types of ionic liquid and lithium salt,p H value,incubation time,microwave irradiation time and power for the remarkably influential factors.The proposed method gave the yields of oligomeric procyanidins(OPC)and polymeric procyanidins(PPC)of 13.01 and32.19 mg/g under the optimal condition liquid-solid ratio 11.91 m L/g,microwave irradiation time 15.95 min and power 581.49 W.The crude procyanidins extracts were purified using HSCCC.Five bioactive fractions were separated under the optimized condition: ratio of solvent system n-hexane-ethylacetate-methanol-water to be 1:40:1:40(V/V/V/V),sample loading amount of 100 mg,separating temperature of 30℃,rotating speed of 950 rpm and flow rate of mobile phase of 2 m L/min.The purity of fractions 1-5 were 96.4%,92.9%,93.4%,95.7% and 96.8%,respectively.The mean degrees of polymerization of fractions 1-5 were 1.64,2.31,4.36,7.89 and 6.94,respectively.The mean molecular weight of fractions 1-5 were 414.75,793.38,1587.96,2476.26 and2176.50,respectively.Fraction 3(F3)was proved to possess the best anti-hepatoma activity and synergistic effect combined with doxorubicin(DOX)in inhibiting the proferation of Hep G2 cells.In order to promote comprehensive utilization and development of procyanidins F3,different drug delivery systems were established.Gold nanoparticles coated liposomes using procyanidins F3(F3-Lips@Au NPs)were prepared.Procyanidins F3 were modified on phosphatidylcholine and acted as reductant,stabilizer and model drug encapsulated inside the hydrophilic core.Under the optimized condition(ultrasound irradiation power 40% amplitude,ultrasound irradiation time 2 min,ratio of HAu Cl4·3H2O and F3 1:1 and reaction time 4 h),F3-Lips@Au NPs with particle size of 350 nm was prepared.The as-prepared material possessed good photothermal property and controlled drug release capacity.Taking doxorubicin(DOX)as the model drug,the proposed material was proved to exhibit synergistic antitumor effect combining with procyanidins F3 after being irradiated by 808 nm laser.Then inspired by the natural cell,red blood cell mimetic microcapsule(RBCM)with better biocompatibility was prepared.This part of thesis was to construct RBCM based on layer-by-layer self-assembly method.RBCM was constructed with PAH,zein and glutaraldehyde as self-assembly materials,magnetic nanoparticles as functional materials.RBCM possessed the features of similar size and biconcave discoidal morphology of real red blood cell.RBCM was propelled with speed of8.80 μm/s by external rotating uniform magnetic field with magnetic field strength of 20 m T and magnetic frequency of 10 Hz.RBCM also kept good motion state in different biological media.RBCM could be used as the negative contrast agent for magnetic resonance imaging.A model of xenograft tumor of Hep G2 in nude mice was established,and it was observed that RBCM with encapsulated procyanidins F3 and DOX possessed long-circulation performance and good anti-hepatoma effect.With crude procyanidins extracted by aqueous enzymatic pretreatment ionic liquid-lithium salt based microwave-assisted extraction method,this thesis further purified procyanidins by HSCCC and made composition analysis of different elution fractions.In order to keep the stability and bioactivity of procyanidins F3,two micro-nanocarriers including gold nanoparticles coated liposomes using procyanidins F3 as well as RBCM with good biocompatibility and biodegradability were constructed.The construction of different multifunctional drug delivery systems could provide technical support to the exploitation of natural products. |
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