Study On Preparation, Identification And Function Of Antioxidant Carrier Based On Phosphatidylcholine

Vitamin c (Vc) is an non-enzyme free radical scavenger that was widely studied. Uptake of Vc by glucose transport decrease when blood glucose increase. Activity of sodium-dependent vitamin C transporter would be inhibited when administrating high dose of Vc. Technology for improving Vc bioavailability need to be develop. Catalase, a typical anti-oxidative enzyme, has too large molecule weight to be absorbed in intestine and is easy to be degraded by protease within gastrointestinal tract and cell lysosome. Carrier should be developed for protecting this enzymes from degradation. Formation of phospholipid complex could increase bioavailability of many active molecules, having potential application to improve Vc bioavailability. Phospholipid based solid lipid nanoparticles (PC-SLN) is a new oral protein/peptide carrier protecting protein from degradation, having potential application in catalase protection. PC of high purity is the major material for preparation of PC-drug complex and PC-SLN. There are no developed thchnology for PC production in China. Macroporous ion exchange resin chromatography is the widely used method in bioactive molecule enrichment, having protential use in large-scale production of PC. This study was aimed to develop PC purification technology based on Macroporous ion exchange resin chromatography, to develop method for preparation of Vc-PC complex and catalase loaded PC-SLN, and to study the their physicochemical property and biologic activity.Firstly, HPLC-UV method was developed for detection of soybean phosphatidylcholine. Analysis conditions were: lichrospher Si (25cm×4.6mm,5μm); mobile phase, A-hexane / isopropanol(v/v)=1:1 and Hexane / isopropanol / 1% acetic acid (v/v/v); Flow rate, 1.0mL/min; column tempreture, 35oC; detection wavelength, 205 nm. PC and phosphatidylethanolamine were well separated within 25 min.S resin was the best macroporous ion exchange resin for PC purification. The optimum conditions for chromatography were: sample concentration, 3 mg/mL (w/v); adsorption velocity, 1 mL/min; eluant, 95% ethanol; elution rate, 1 mL/min. Product was powder PC of light yellow (94.02%, wt%), and the yield was 80.74%. Pattern for adsorption of soybean PC onto S resin were investigated, suggesting that the adsorption kinetics of PC on S resin fitted well with shell-core model and the adsorptive processes were controlled by pore diffusion of adsorption layer. Adsorption isotherm model was fitted with preferential adsorption isotherm and Langmiur equation. Adsorption equilibrium reached at 240 min. Adsorption reaction is exothermic and high temperature is not conducive to the adsorption.Complex of soybean PC and Vc (Vc-PC) was preparation and physicochemical properties were studied. Ethanol and chloroform was served as reaction and separation solvent, respectively. Optimum reaction conditions was: molar ratio of Vc:PC, 1:1; reaction time, 3 h, the reaction temperature, 50oC, Vc concentration, 0.1 mg/mL. Vc and phospholipid in the complex were joined by non-covalent-bonds and did not form a new compound, revealed by infrared spectrometry (IR), differential scanning calorimetry (DSC) and atomic force microscope study. Vc-PC had good solubility in aprotic solvent. Mice peritoneal macrophages were prepared and oxidative stress was induced with LPS. Antioxidative effects were studied for different concentrations of Vc and Vc-PC. Supernatant NO, lactate dehydrogenase (LDH), malondialdehyde (MDA) and Intracellular vitamin C, inducible NO synthase (iNOS) was measured. Vc-PC significantly increased intracellular Vc and inhibited cell lysis in LPS induced cell. This biological effect was more effective than that of Vc, demonstrating that PC-Vc enter cell by the transporter independent manner. Vc-PC might be effective for Vc uptake without the influence of glucose and down-regulation of Vc transporter in high dose of Vc treatment.Catalase loaded solid lipid nanoparticle (CAT-SLN) was prepared by the double emulsion method (W/O/W) and solvent evaporation technique, using acetone/methylene chloride (1:1) as an organic solvent, soy bean PC and tripalmitin as oil phase and Poloxmer 188 as a surfactant. For optimized precedure, catalase solution (20 mg/mL) (internal water phase) was infused into dichloromethane/acetone (1:1) containing tripalmitin (TP) and soybean PC (PC:TP =15.24%)(oil phase), internal water phase:oil phase=5:1(v:v). Primary W/O emulsification was formed after 20 s of sonication and was infused into 1% Poloxmer 188 (2nd phase). W/O: 2nd phase=1:4 (v:v). After 30 s sonication, W/O/W emulsification was formed. The organic solvent was evaporated by rotary evaporation. Nanoparticles were recovered by centrifugation. The CAT-SLN was spheroidal and had homogeneous size. Its Z potential was -37.1 mV, and polydisperse index was less than 3. Catalase was mainly distributed in the inner phase of the SLNs, realsing slowly from SLN without change of structure. Catalase loaded in SLN was protected against proteolysis.This study provided scientific base for development of new type food free radical scavenger, to prevent chronic disease.