酚酸-透明质酸钠接枝物的制备及体外抗氧化研究

Preparation of Phenolic Acid-sodium Hyaluronate Copolymers and in vitro Antioxidant Activity Assessment

目的以透明质酸钠(sodium hyaluronate,HA)为研究对象,利用酚酸对其修饰改性以期获得抗氧化活性更好甚至新活性的分子结构。方法采用自由基介导的接枝方法制备5种不同的酚酸-透明质酸钠接枝物,选出接枝度最高的接枝物进行合成条件优化。通过红外光谱(infrared spectroscopy,IR)、紫外光谱(ultraviolet spectrum,UV)、核磁共振氢谱(^(1)H NMR spectroscopy,^(1)H NMR)、场发射扫描电镜(field emission scanning electron microscopy,FESEM)、热重分析法(thermogravimetry analysis,TGA)对接枝物进行表征,通过测定接枝物对1,1-二苯基-2-三硝基苯肼(DPPH·)、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS^(+)·)及超氧阴离子自由基(O^(2-)·)清除能力反应出接枝物的体外抗氧化能力。结果在相同条件下,5种酚酸-透明质酸钠接枝物种中接枝度最高的是阿魏酸-透明质酸钠接枝物(ferulic acid-sodium hyaluronate FA-HA),对FA-HA合成条件行进优化,得到最高的接枝度为(16.59±0.31)mg/g。通过IR、UV、^(1)H NMR、FESEM、TGA验证了接枝物的形成,推测可能形成了酯键。分子质量检测结果表明,通过本方法得到的接枝物的分子质量有小幅下降但分子分布均一性提高,平均分子质量为31.5 ku。FESEM结果显示,接枝物呈现紧密相连的片状结构,表面相对光滑。热重分析(TGA)结果表明,接枝物的热稳定性降低。体外抗氧化实验结果表明,在0.25~10 g/L浓度范围内,FA-HA对DPPH·、ABTS^(+)·和O^(2-)·的最大清除率分别为(83.76±4.86)%、(76.95±5.06)%和(83.08±2.51)%,高于原HA及FA。结论自由基接枝法成功将FA与HA接枝在一起,且接枝后的FA-HA具有更好的体外抗氧化活性,为酚酸-HA接枝物的深入研究和开发应用提供了理论依据。

Objective Sodium hyaluronate(HA)was used as the research object to modify it with phenolic acid in order to obtain the molecular structure with better antioxidant activity or even new activity.Methods In this study,5 kinds of phenolic acid-sodium hyaluronate was prepared by free radical-mediated grafting method,and the grafts with the highest grafting degree were selected to optimize the synthesis conditions.Then,grafts structure and physicochemical properties were analyzed.The grafts were characterized by IR,UV,^(1)H NMR,FESEM and TGA spectra.The in vitro antioxidant capacity of grafts was determined by the scavenging ability of DPPH·,ABTS^(+)·and O^(2-)·.Results Among 5 kinds of phenolic acid-sodium hyaluronate,the grafting rate of ferulic acid-sodium hyaluronate copolymer(FA-HA)was highest,which was chosen as experimental sample in the following tests.Firstly,the reaction conditions were investigated and the highest grafting rate was(16.59±0.31)mg/g at the optimal preparation conditions.Then,FA-HA structure and physicochemical properties were analyzed.Data from UV,IR,^(1)H NMR analyses,TGA showed that FA were successfully grafted to HA.Compared with HA,the results of gel permeation chrematography(GPC)showed that the molecular mass distribution of FA-HA copolymer decreased from 34.4 to 31.5 ku,but the uniformity of molecular distribution was improved.FESEM results showed that the structure of copolymer exhibited a closely connected lamellar structure with a relatively smooth surface.TGA results showed that thermal stability of FA-HA had a little decline.The antioxidant performance in vitro results showed that,during 0.25-10 g/L,FA-HA can eliminate(83.76±4.86)%DPPH·,(76.95±5.06)%ABTS^(+)·and(83.08±2.51)%O^(2-)·respectively at 10 g/L.which were higher than that of native HA and FA.Conclusion FA and HA were successfully grafted together by free radical grafting,and the grafted FA-HA had better antioxidant activity in vitro,which provided a theoretical basis for further research and development of phenolic acid-HA grafts.