Study Of The Enzymatic Synthesis Of Galactosyl-Glycerol-Lipids And The Anti-Tumor Activity

Glyceroglycolipid is a natural product that could find interesting applications in foodstuffs, cosmetic and health-care products. It was also shown in several studies to have anti-viral, anti-tumor, anti-bacterial, anti-inflammatory, antioxidant and other biological activity. However, by extraction method, the purity and the content of glyceroglycolipids were limited; chemical method or enzymatic-chemistry method was with complicated steps, harsh conditions, poor stereoselectivity and regioselectivity. So it’s difficult to accurately reflect the structure and activity relationship of glycoglycerolipid. The aim of this study is to develope a method of enzymatic synthesis of galactosyl-glycerol-lipids, and then separation, structure characterisation and anti-tumor activity are also investigated. The main contents are as follows:1. Study of an enzymatic method for the production of galactosylglycerol is described in this chapter. The P-Galactosidase from Kluyveromyces lactis was selected as the catalyst and the effects of enzyme concentration, reaction temperature, reaction time, substrate ratio of glycerol to galactose, buffer content and pH on galactosylglycerol yield (mg/mL) were investigated. Based on the results of one-factor-one-time experiments, response surface method was used to optimize the reaction conditions (substrate ratio, enzyme concentration, reaction temperature and reaction time). By analyzing the response surface plots and the quadratic regression equations, the results indicated that the optimal conditions were as follows: temperature 39.75℃, time 48 h, enzyme concentration 350 U/mL, and the substrate molar ratio 8.65:1 (glycerol:galactose), the yield of galactosylglycerol could reach 140.03±5.87 mg/mL, and the conversion could reach 55.71±2.33%.This result was close to the predicted values, and higher 20.22% than a single factor experiments (116.47±3.56 mg/mL).2. Galactosylglycerol was synthesized from glycerol and galactose catalyzed by (3-galactosidase. Activated carbon adsorption method was used to separate and purify product. Static adsorption tests indicated that the adsorbed concentration was 56.77 mg/mL, equilibrium adsorption time was 1h, the temperature was 20℃.The kinetic data were respectivitely fitted by pseudo-first-order equation,pseudo-second-order equation and the intra-particle diffusion model. It was found that the pseudo-second-order equation could provide the best correlation to the data. The adsorption activation energy Ea was 62.58 kJ/mol, indicating the adsorption process belonged to the physical adsorption. According to the results of thermodynamics, the adsorption process was spontaneous and exothermal (ΔG<0, ΔS>0), and the entropy is negative (ΔH<0). The adsorbing and eluting process were also studied by dynamic method.Under the conditions of sample concentration 21.46 mg/mL and flow rate 2mL/min, and 3% alcohol-water was used to elute glycerol and galactose, then 9% alcohol-water was used to obtain the galactosylglycerol. Purity of the final product was 98.33%, and the recovery rate was 76.95%. The purified galactosylglycerol was identified as a (1:1) mixture of (2R) and (2S)-3-O-β-D-galactopyranosyl-sn-glycerol by the combined used of 1D and 2D NMR experiments. MS analysis of galactosylglycerol gave molecular weight at m/z 254. The resulting product can be used for the synthesis of galactosyl glycerides.3. Lipase-catalyzed transesterification of galactosylglycerol and hexanoic acid in nonhydrous medium was investigated in this chapter. The Novozyme 435 was selected as the catalyst and the effects of enzyme concentration, reaction temperature, reaction time, substrate molar ratio on the conversion of galactosylglycerolipid were investigated. The conditions were as follows:Novozyme 435 as a catalyst, acetone as the medium,20 mg/mL of enzyme concentration,1:18 of substrate molar ratio (galactosylglycerol to hexanoic acid), at 50℃ for 20.5 h. Under these conditions, the conversion of galactosylglycerolipid was up to 86.82%. The separation of products was completed by the solvent extraction and silica gel chromatography. The monohexanoyl-galactosylglycerol in the water phase were eluted by chloroform: methanol (v:v 9:1) and the dihexanoyl-galactosylglycerol in the chloroform phase was eluted by chloroform:methanol (v:v 9:1). The purified products was fully characterised by MS and NMR, and the monohexanoyl-galactosylglycerol was identified as a mixture of 1-O-hexanoyl-3-O-β-D-galactopyranosyl-glycerol and 3-O-(6’-O-hexanoyl-β-D-galac topyranosyl)-sn-glycerol, with m/z 352; the dihexanoyl-galactosylglycerol was identified as 1-O-hexanoyl-3-O-(6’-O-hexanoyl-β-D-galactopyranosyl)-sn-glycerol, with m/z 450.4. We synthesized various mono- and di- galactosylglycerolipids with different acyl chains (C6, C10, C12, C16, C18, C18:1, C18:2, C18:3), and tested their anti-tumor activity in this chapter. All products were detected by HPLC-ELSD and MS identification. Purity of all pruducts was above 95%. Galactosylglycerolipids exhibit inhibition effects on the cancer cells A549, BGC-823, HepG2, MCF-7 and normal cell 293T. Experimental results show that the acyl chain of galactosylglycerolipids plays an important role in the anti-tumor activity. Among the products that were synthesized above,mono-/di-decanoyl-galactosylglycerolon, and mono-/di-palmitoyl-galactosylglycerol were selected as potential anti-tumor drugs. The mechanism of these products need further study.