基于柱前衍生-HPLC法研究焦栀子炮制过程单糖组成及含量变化

Composition and content change of monosaccharide in Gardeniae FructusPraeparatusduring heat processing based on pre-column derivatization HPLC method

目的建立柱前衍生-HPLC法测定焦栀子炮制过程样品中8种单糖(甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖)的含量测定方法,通过分析比较总单糖、结合型单糖和游离单糖的组成及差异,为揭示焦栀子炒制过程颜色褐变机制奠定基础。方法以Phenomenex Luna C18柱(250 mm×4.6 mm,5μm)为色谱柱,流动相为100 mmol/L乙酸铵-乙腈(83∶17),柱温35℃,体积流量1.0 mL/min的条件下,分别对酸水解糖提取方法、游离糖提取条件进行优化,衍生化后样品进行方法学考察。结果酸水解方法以1.5 mol/L硫酸、100℃水解4.0 h最佳,游离单糖为超声处理15 min。焦栀子炮制过程样品酸水解产物检测出甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、木糖、阿拉伯糖共8种单糖;游离单糖检测出甘露糖、葡萄糖和半乳糖。经方法学验证,8种单糖的精密度、重复性、稳定性、加样回收率均符合方法学要求;酸水解得到的总单糖在种类上、含量上均高于游离单糖,总单糖和结合型单糖部分糖变化基本一致,甘露糖含量呈先降后升的趋势,半乳糖醛酸含量呈降低趋势,总单糖葡萄糖含量呈先升后降趋势,结合型葡萄糖含量呈升高的趋势,其他糖的变化幅度不大。随着炒制程度的加重,过程饮片的结合型单糖的组成类别不变,除结合型半乳糖醛酸外,各结合型单糖比例基本不变。游离葡萄糖和游离半乳糖含量随炮制程度的加深呈降低的趋势,其中游离半乳糖在焦栀子中未检出。结论游离葡萄糖、游离半乳糖和结合型半乳糖醛酸是焦栀子炮制过程中变化最明显的单糖,可为栀子炒焦过程颜色褐变机制的反应物提供参考。

ObjectiveReversed-phase-high-performance liquid chromatography(RP-HPLC)precolumnderivatization was used to determine the monosaccharide composition and content in Jiaozhizi(Gardeniae FructusPraeparatus,GFP)during heat processing.By analyzing and comparing the composition and difference of total monosaccharides,binding monosaccharides and free monosaccharides,the basis was laid for revealing the mechanism of color browning during the heating process of GFP.Methods Chromatographic separation was carried out on a Phenomenex Luna C_(18)column(250 mm×4.6 mm,5μm)with the mobile phase of 100 mmol/L ammonium acetate-acetonitrile(83:17)at the flow rate of 1.0 mL/minand the column temperature of 35℃.Sample pretreatment conditions were optimized by investigating the effect of extraction solvents and methods on the extraction efficiency of monosaccharide used for HPLC quantification.Results Total monosaccharides was measured after hydrolysis in 1.5 mol/L sulphuric acid at 100℃for 4.0 h and free monosaccharide was measured after ultrasonic in water for 15 min.Eight total monosaccharides,including mannose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,xylose and arabinose,athree free monosaccharides,including mannose,glucose and galactose,were detected in the GFPduring heat processing.The developed HPLC method for the determination of eight monosaccharides was validated in terms of linearity,precision,reproducibility,stability,and recovery of the eight monosaccharide.The total monosaccharides obtained by acid hydrolysis was higher than free monosaccharides in terms of variety and content.The change of total monosaccharide and some sugars of conjugated monosaccharides were basically the same.The mannose content showed a trend of decreasing first and then increasing,the galacturonic acid content showed a trend of decreasing,the total monosaccharose glucose content showed a trend of increasingfirst and then decreasing,the bound glucose content showed a trend of increasing,other sugars changed little.With the aggravation of the stir-frying degree,the composition of the bound monosaccharides of the processed slices remained unchanged,except for the bound galacturonic acid,the proportion of the bound monosaccharides remained basically unchanged.The content of free glucose and galactose decreased with the deepening of processing,and free galactose was not detected in GFP.ConclusionFree glucose,free galactose and bound galacturonic acid were the most obvious monosaccharide in the heat processing of GFP,which canprovide clues for the reaction substances of the color browning mechanism of GFP.