| Lipid from Ginkgo biloba leave(GL)is a kind of important and fat-soluble bioactive natural product in the Ginkgo biloba leave(GBL),in which polyprenols(PPs)has the similar structure with dolichol,and possesses the function of preventing hypertension,resisting tumour,protecting liver,curing senile dementia and so on,but it has not been developed and utilized yet.Therefore,in this paper,cell wall disruption assisted-extraction technologies were used to extract GL,and new materials were prepared to purify GL for obtaining PPs with high purity.In addition,GL lipidosome and gels were prepared and their biological activities were also investigated.These researches could provide some theoretical references for developing new pharmaceuticals,functional food and cosmetic.The main contents and results are as follows:(1)Process optimization of extracting GL using cell wall disruption technologiesComparing effects of extracting GL between enzymolysis-assisted ultrasound method and mechanochemical method,and optimizing their extraction process.The optimum process conditions of extracting GL by enzymolysis-assisted ultrasound was an enzyme quantity of 0.5g(the mass ratio of cellulase and pectinase was 1:2,and the enzyme activity was 60 U/mg),enzymolysis pH of 4.5,and temperature of ultrasound of 45℃.Under these conditions,the yield of PPs could reach 0.80%.The optimum process conditions of extracting GL by mechanochemical method was an concentration of NaOH of 30%,solid-to-liquid ratio of 1:15,extraction temperature of 80℃,and extraction time of 4 h.Under these conditions,the yield of PPs was 0.81%.Therefore,both enzymolysis-assisted ultrasound extraction method and mechanochemical extraction method could increase the yield of PPs,but considering alkali liquor produced from mechanochemical extraction method was not easy to be processed,so enzymolysis-assisted ultrasound extraction method was selected to extract GL in the future.(2)Separation and functional characteristics of water soluble ingredients in the enzymatic hydrolysatePolysaccharide and protein were main ingredients in the enzymatic hydrolysate.Absolutealcohol was used to precipitate polysaccharides in the enzymatic hydrolysate,then three polysaccharide monomers GBP11,GBP22 and GBP33 were achieved by DEAE cellulose column chromatography and G-100 gel column chromatography,the molecular weight of which were 1.94×103 kDa,5.17×103 kDa and 46.0 kDa,respectively.Moreover,these three kinds of polysaccharide monomers were all mainly made up of glucose,galactose,arabinose and rhamnose,the molar ratio of which were 62.2:2.0:3.8:1.2,3.6:68.0:4.7:0.4 and5.3:48.8:1.7:9.0,severally.Chitosan was used to precipitate protein in the enzymatic hydrolysate,then protein with purity of 90.4% was obtained by actived carbon decoloration and dialysis,and the highly purified protein possessed good solubility,water(oil)binding capacity as well as emulsibility and emulsion stability.(3)Change rule of physicochemical properties for the GBL before and after cell wall disruption technologies assisted extractionPyrolysis-gas chromatography/mass chromatography(Py-GC-MS),Scanning electron microscope(SEM),thermogravimetry(TG)and high performance liquid chromatography(HPLC)technologies were utilized to research change rule of physicochemical properties for the GBL before and after cell wall disruption technologies assisted extraction,including content of PPs,appearance of cell wall,pyrolysis products,thermal stability and so on.The results of SEM showed that the cell wall surface of untreated GBL was smooth,and arrange among the adjacent hole was dense.While the cell wall surface of GBL treated with cell wall disruption technologies was seriously destroyed,a number of holes appeared,and arrange among the adjacent hole was loose.The results of TG indicated that GBL treated with cell wall disruption technologies were easier to decompose compared with untreated GBL.In addition,the thermal decomposition mechanism of GBL before and after cell wall disruption technologies assisted extraction were all random nucleation and growth reactivity control mechanisms,according with Avrami-Erofeev equation.The results of HPLC revealed that there was almost no PPs existing in the GBL after cell wall disruption technologies assisted extraction.The results of Py-GC-MS demonstrated that small molecule compounds which less than 12 carbon atom were mainly composed of water-soluble ingredients in the GBL after cellwall disruption technologies assisted extraction,and its total contents of GC obviously decreased after cell wall disruption technologies assisted extraction.Total contents of GC of Low polarity components with number of carbon atom between 12 and 20 were variational based on different cell wall disruption technologies.(4)Synthesis and characterization of materials used for purifying GLNew nano-silica materials modified by sulfydryl or ionic liquid as well as silver ion were synthesized and characterized.GL was separated and purified by using the new materials,while adsorption kinetics,adsorption isotherm and adsorption mechanism were investigated in the process of separation and purification.The results of IR showed that sulfydryl and ionic liquid had successfully fixed to the nano-silica because of no characteristic adsorption peak of hydroxy appeared.The results of SEM manifested that agglomeration phenomenon of the new materials became weaken,and dispersibility among particle were better.The results of XRD proved that chemical modification had some influences on the structure of nano-silica,but the influence was little.The results of adsorption kinetics indicated that the adsorption of GL on the new material reached equilibrium at the time of 7 minute,the adsorbing capacity of which was 83.4 mg/g,conforming to second order kinetics.Using new material as fixed bed to purify GL,the content of PPs increased from 43.9% to 80.8%.The results of adsorption isotherm revealed that the adsorption of GL on the new material was exothermic reaction,and low temperature was in favour of increasing adsorbing capacity.Furthermore,the adsorption of PPs was better than the adsorption of GL on the new material.(5)Thermal decomposition characteristic and mechanism of GLThe thermal stability,breakage situation of chemical bonds,thermal decomposition kinetics and mechanism of GL and PPs standard were researched.The experimental results illustrated that GL had good stability,it started to sharply decompose after 268.3℃,and the contents of methyl,methylene,dissociative alcohol or phenol,aromatic compounds,olefin and ester continually increased with the reaction going on.However,as the irregular of GL thermal decomposition,it was unable to analysis the thermal decomposition kinetics and mechanism of GL.The stability of PPs standard was more stable than GL,the mass of which decreasedslowly before 347.4 ℃,it started to sharply decompose after 347.4 ℃,and the contents of methyl,CO2,unsaturated carbon hydrogen bond,alkenyl,accumulated carbon-carbon double bond,carbon-carbon double bond and hydroxy constantly augmented.Besides,the thermal decomposition process of PPs standard belonged to first order chemical reaction,the thermal decomposition kinetics expression was dα/dT=2.231×1013e(-20712/T)(1-α)/β.(6)Preparation and characterization of GL nano-silver preparationsFilm dispersion method was used to prepared GL lipidosome and gels.The optimum process conditions of preparing GL lipidosome was that lecithin:cholesterol(m:m)=3:1,blank lipidosome: GL solution(m:v)=10:1,ultrasound time was 30 minute.The optimum process conditions of preparing PPs lipidosome was that lecithin:cholesterol(m:m)=3:1,PPs lipidosome: PPs solution(m:v)=5:1,ultrasound time was 30 minute.On this basis,nano-silver GL lipidosome,nano-silver PPs lipidosome,nano-silver GL gels and nano-silver PPs gels were prepared,and the mean grain size of which were 397 nm,1114 nm,3223 nm and 5940 nm,respectively.Physical stability of prepared lipidosome and gels were all excellent,and storage stability of which were better than corresponding solution.(7)Biological activities of GLand its preparationsAntioxidant activities of different polar fractions separated from GL were studied,and bacteriostatic activities of preparednano-silver GL lipidosome and gels were also researched.The results of antioxidant experiments stated that concentration C had strong scavenging activity on the DPPH,and fraction 3 demonstrated powerful scavenging activity on the ABTS and hydroxyl radical,while PPs standard displayed intense scavenging activity on the superoxide anion.The results of bacteriostatic experiment declared that four kinds of prepared nano-silver lipidosome and gels possessed certain inhibitory effects on the escherichia coli,candida albicans,staphylococcus aureus and streptococcus pneumoniae,in which escherichia coli was best inhibited,then were staphylococcus aureus,streptococcus pneumoniae and candida albicans,respectively.Nano-silver GL gels possessed greatest antibacterial effects on the escherichia coli,candida albicans,staphylococcus aureus and streptococcus pneumoniae,and the range of inhibition zone for the nano-silver GL gels was 12.0~21.4 mm,while therange of MBC and MIC values were 0.5~64 μg/mL and 16~256 μg/mL,severally.Furthermore,bacteriostatic activities of nano-silver GL lipidosome and gels were stronger than the nano-silver PPs lipidosome and gels,this may be connected with the synergistic antibacterial effects between non-PPs ingredients and PPs. |
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