Study On The Enzymatic Synthesis Of Phytosterol Lipoate And Their Biological Activities

Lipoic acid is a natural disulfide compound,which is known as a"universal antioxidant"because of its good antioxidant activity.However,lipoic acid has poor lipid solubility,is unstable to light and heat,has a short half-life in vivo,has a strong"first pass effect",and has low bioavailability.These characteristics greatly limit its practical application.Phytosterols are a class of natural hydrophobic components with hypocholesterolemic activity.In this study,lipophilic lipoic acid esters namely phytosterol lipoate was synthesized with phytosterols as hydrophobic modifiers using lipase-catalyzed esterification in non-aqueous media.On this basis,its antioxidant activity in different food systems and its protective effect on high-fat diet-induced nonalcoholic fatty liver disease(NAFLD)were investigated.The main findings were as follows.1.Enzymatic synthesis,separation and purification,structural characterization and process optimization of the products.Different lipases were used to catalyze the esterification of lipoic acid with sitosterol,and the product was confirmed as the target product,lipoic acid sterol ester,by mass spectrometry,Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy.The most suitable process parameters were obtained as follows:25 g/L Candida rugosa lipase,isooctane,100 mmol/Lβ-sitosterol,100 mmol/L lipoic acid,120 g/L 3?molecular sieve,40℃,18 h.Under these conditions,the conversion of the product reached 85.7%,and the product was separated by column chromatography.The product yield was 74.9%.Compared with lipoic acid and sitosterol,the solubility of lipoic acid sterol ester in vegetable oil was increased at least 26.7-fold and 2.9-fold,respectively,indicating that the lipid solubility of lipoic acid was significantly enhanced after esterification with phytosterols.2.Antioxidant activity of lipoic sterol esters.The melting point of lipoic sterol esters and the effect on the melting characteristics of oils and fats were investigated.The results showed that the melting point of lipoic acid sterol ester was significantly lower than that of sitosterol by 97℃,and its addition to vegetable oil did not affect the melting characteristics of vegetable oil.The reducing power,DPPH radical scavenging ability,and antioxidant activity of lipoic acid sterol esters in oil&fat system,emulsion system and cooked minced meat system were further investigated.The results showed that the reducing power and DPPH radical scavenging ability of lipoic acid sterol ester were better than those of lipoic acid and sitosterol,and the scavenging ability of DPPH radicals was positively correlated with the concentration,and its DPPH radical scavenging ability was comparable to that of BHT at low concentration.Lipoic acid sterol esters can effectively slow down the oxidation of oils&fats,mainly by inhibiting the heating-induced increase of peroxide value(58.8%),anisidine value(42.8%)and total oxidation value(54.9%)of oils&fats,and the degradation of polyunsaturated fatty acids(16.1%).The inhibitory ability of lipoic acid sterol ester on oil oxidation is comparable to BHT and significantly better than lipoic acid and sitosterol.In addition,the sterol ester of lipoic acid showed good antioxidant activity in theβ-carotene-linoleic acid system,β-carotene-AAPH system and cooked minced meat system,and was superior to lipoic acid and sitosterol.3.Protective effect of lipoic acid sterol esters on high-fat diet-induced NAFLD in mice.The effects of lipoic acid sterol esters on high-fat-induced hepatic lipid aggregation,plasma lipids and fecal lipids,and plasma and hepatic antioxidant parameters were investigated.The results showed that sterol lipoic acid esters could effectively prevent NAFLD by improving disorders of lipid metabolism in mice,mainly by significantly reducing perirenal fat coefficient(62.2%),epididymal fat coefficient(55.0%),(perirenal+epididymal)fat coefficient(58.1%),total plasma cholesterol(16.7%),triglyceride(22.2%),non-HDL cholesterol(23.8%)and non-HDL cholesterol/HDL cholesterol ratio(22.5%),liver cholesterol(50%)and fatty acid content(45.8%),and increased fecal fatty acid content(12.2%),respectively.In addition,lipoic acid sterol esters were effective in improving liver tissue morphology and reducing the formation of fat droplets.In addition,sterol lipoic acid esters could also atteunate NAFLD by improving oxidative stress,mainly by significantly increasing plasma total antioxidant capacity(52.4%),catalase activity(66.2%),liver total antioxidant capacity(75%),superoxide dismutase activity(46.8%)and catalase activity(21.9%),and significantly decreasing plasma(43.6%)and liver(47.9%)malondialdehyde levels.In contrast,the amelioration of abnormal lipid metabolism and oxidative stress induced by high-fat diet by lipoic acid sterol ester was significantly better than the physical mixture of sitosterol and lipoic acid,indicating that the protective effect of lipoic acid sterol ester on NAFLD was stronger than that of the physical mixture of sitosterol and lipoic acid.