| The preliminary studies, which were based on the study of forming active matter carrier system between the food grade bioactive peptides-casein phosphopeptides(CPP) and the active polysaccharide-chitosan(CS) systermatically study the interaction relationship between CPP and CS, have found out the best conditions and steps for the formation of nanoparticles. This paper attempt to embed a natural polyphenol-Oenothein B(OeB) by using CPP/CS. However, as a kind of macrocyclic two-polymer tannin, OeB is easy to interact with CPP and CS, which changes the intrinsic properties of CPP and CS. Consequently, in order to embed OeB effectively, this paper studied on the influencing factors of the interaction among CPP, CS and OeB.The study is based on establishing a three-phase transition diagram and its mechanism model, and guiding the production of OeB nanoparticles theoretically. In addition, a suitable preparation method for industrial production is proposed by means of optimizing the technology and making a comprehensive evaluation from stability, slow-release effect and absorption effect of Nano ball. The follows list the main research contents and results:(1) Study on the interaction of CPP/CS/OeB. By changing the ratio and concentration, obsevering the phenomena and collecting the characteristic parameters including Particle Size(PZ), Count Rate(CR), Polydispersity(PD) and Turbidity(T), the three-phase transition diagrams were drawn out. The following rules were summarized by the experiments: In the system of CS/OeB, when the concentration is below 0.9mg/m L, the system shows clear and colorless. When the concentration is over 1.8mg/m L,with the increase of the proportion of OeB and CS, the turbidity and millivolt value of CS/OeB system increase, with the change of Solution-Sol-Flocculation. In the system of CPP/CS, the system goes through the change of Solution-Complex Coacervate-Precipitation with the increase of CPP and CS quality ratio whatever the concentration is, when the quality ratio of CPP and CS is greater than 3, there is particle Precipitation generating in the system. Within the scope of the concentration of 3.0 mg/m L, the combination of proportion, forming particle size small(222.9~312.7 nm), light intensity value(429.8 ~ 621 KCPS) polyelectrolyte nano ball, on the other hand, with the increase of content of CS, the light intensity value is on the decline; When both scale unchanged, with elevated levels of total particle size parameters, light intensity value, turbidity are on the rise. In CPP/OeB system, regardless of the change of concentration, both interaction phenomenon is not obvious, system will remain clear. In the CPP/CS/OeB system, there are four state:(1)Solution,(2)Complex Coacervate,(3)Flocculation,(4)Precipitation. Based on the above four kinds of phase state and the proportion of CPP/CS/OeB, four phase diagrams of different concentrations were drawn out in the triangle coordinates. The comparison of the phase transition diagrams showed that the main factor affecting the three-phase transitions was proportion, and the second was concentration. Considering from the particle size and number of the polymer CPP/CS/OeB and system stability etc., the polyelectrolyte nanoparticle whose final concentration of the system was 1.4mg/m L and the ratio of CPP/CS/OeB was 3:3:1, was selected for the further processing. And this nanoparticle's particle size was 156.1±2.2 nm, Zeta was 22.02±1.3 m V, CR was 652.1 kcps, and polydispersity was 0.124.(2)Optimization of preparation technology of OeB nanoparticles. Nanoparticles were processed by four techniques, including cross-linking, centrifugation, ultrasound, and freeze drying. By changing the natural crosslinking agent Genipin's additive amount and time of action, the particle size, CR, encapsulation efficiency and drug loading of OeB nanoparticles were studied under the different p H conditions before and after crosslinking. OeB crosslinked nanoparticles were enriched by the method of high speed centrifugation, and its changes of particle size and CR in supernatant were used to evaluate the effect of separation and enrichment in different relative centrifugal and centrifugal time. Ultrasonic technology is beneficial to disperse nanoparticles Precipitation. For analyzing the dispersion effect, the study compared the changes of polydispersity, particle size, CR and turbidity by changing the ultrasonic time and power. The freeze drying technology keeps the characters of the nanoparticles, which is convenient for storage, transportation and industrial production, and the effect of the freeze drying process is evaluated by scanning electron microscope. The results of the study showed that when the concentration of Genipin was 0.4 mg/m L and the crosslinking time was up to 12 h, the encapsulation efficiency of OeB was 99% and the drug loading was 15%, and when the p H was reduced to 2, the particle size and the CR of OeB crosslinked nanoparticles had no obvious change; When the centrifugal condition was 13,400 g and 30 min, the CR in supernatant was redused from the 652.1 kcps to 60.2 kcps; After 45 min treatment in 100 W power, the polydispersity decreased from 0.319 to 0.208, and the particle size decreased from 339.9 nm to 208.1 nm. According to the scanning electron microscope, the size distribution of OeB crosslinked nanoparticles was mainly in 50~100 nm, and a small amount was distributed in the range of the 200~500 nm. The parameters of OeB crosslinked nanoparticles after optimized were as follows: the average particle size was 210±50 nm, Count Rate range was 550~650 kcps, Zeta was 18.06±1.8 m V, and polydispersity was 0.200 ±0.05.(3)The controlled and released effect and stability evaluation of OeB-GNP. First, the effects of p H, temperature, time and enzyme solution environment on the stability of OeB and the effect of OeB-GNP controlled release were studied by the retention rate of OeB and the release rate of OeB in the nano spheres; Secondly, with the particle size, light intensity, dispersion coefficient and turbidity as index to study the influence of p H, temperature, storage time and solution environment on nano ball traits; Finally, using the fluorescence value as index, using eliasa capability analysis of OeB-GNP scavenging activity and fluorescence microscopy instrument. The results showed that, at p H1.2~7.0, 4~25 ? temperature conditions, OeB stable retention rate was(99±1)%; In addition, there was no OeB released from the OeB-GNP. In the four kinds of enzyme liquid, at 6h after OEB retention rate from small to large followed by pancreatic lipase Solution < alpha-amylase liquid < artificial simulation intestinal < artificial simulated gastric fluid, retention rates were(6.2 ± 0.6)%,(7.2 ± 0.8)%,(36.7 ± 1.4)%,(56.7 ± 0.6)%; OeB release rates changed at different time, but all of them are less than 10%, which in artificial gastrin releasing lowest rates, 0.2 to 0.6%, the highest release rate in artificial gastric juice, 0.4 to 9.6%.OeB-GNP showed stability at p H 1.2~6.5 conditions, as well as different Solution conditions, the particle size in the range of 172.9~280.5 nm, light intensity in the range of 405.3~600.0 kcps, fluctuated within a reasonable range; At different temperatures, the lower OeB-GNP storage temperature, the longer the storage time of the ball, under the condition of 4?, OeB-GNP traits remained stable at 20 days. Cell antioxidant capacity of the study showed that OeB-GNP has a certain antioxidant activity, and showed a good dose effect relationship, when the CAA value reached 50, OeB in the mass concentration of 0.117mg/m L OeB-GNP.(4)Evaluation on the absorption effect of OeB-GN. Cell model of Caco-2 is successfully established this paper, investigating reaction of OeB-GNP uptake and transport characteristics of single cells in Caco-2, evaluating its absorption effect in the intestinal. Using inverted fluorescence microscope to observe the result of the uptake of Caco-2 cells experiment that the OeB crosslinked nanoparticles could be attached to the surface of Caco-2 cells. And using method of HPLC to detect OeB from BL after OeB or OeB-GNP transmembrane, failed to detect OeB. The characterization parameters of BL side transfer Solution detected by Dynamic light scattering method shows that with the time extending and concentration increasing of transport, the Count Rate values of BL side transfer Solution is enhanced, particle size distribution is mainly in the range of 80 ~105 nm, it indicates that OeB-GNP pass through the cell model in integral. |
PDF Full Text Request