不同产地苍术低聚糖的糖谱特征分析

Analysis of Oligosaccharide Mapping of Atractylodis Rhizoma from Different Habitats

目的:建立基于超高效液相色谱串联四极杆飞行时间质谱法(UPLC-Q-TOF-MS)的苍术低聚糖快速分析方法和基于超高效液相色谱-蒸发光散射检测器法(UPLC-ELSD)的苍术低聚糖定量方法,考察不同产地苍术药材的低聚糖糖谱特征。方法:通过UPLC-Q-TOF-MS对苍术的低聚糖成分进行鉴定,流动相为0.1%氨水乙腈溶液(A)-0.1%氨水溶液(B)梯度洗脱(0~0.5 min,98%A;0.5~2.0 min,98%~89%A;2.0~2.5 min,89%~86%A;2.5~5.5 min,86%~80%A;5.5~6.5 min,80%~72%A;6.5~9.5 min,72%~63%A;9.5~14.0 min,63%~50%A;14.0~16.0 min,50%A;16.0~16.5 min,50%~98%A;16.5~20 min,98%A),柱温60℃,流速0.2 mL·min-1,电喷雾离子源(ESI),负离子模式下采集数据,扫描范围50~1 500。通过保留时间、相对分子质量、一级和二级MS特征碎片离子信息,并结合对照品信息完成低聚糖的定性分析。采用UPLC-ELSD对苍术中9种低聚糖进行定量测定,流动相0.1%氨水乙腈溶液(A)-0.1%氨水溶液(B)梯度洗脱(0~1 min,98%~75%A;1~7 min,75%~70%A;7~18 min,70%~55%A;18~23 min,55%A;23~23.5 min,55%~98%A;23.5~28 min,98%A),ELSD的漂移管温度50℃。采用主成分分析(PCA)及正交偏最小二乘法-判别分析(OPLS-DA)综合评价不同产地苍术的分组趋势及差异性低聚糖成分。结果:从苍术中共鉴定了24个低聚糖成分,含11对同分异构体。其中,与安徽、重庆、江苏南京、陕西比较,道地产区茅山苍术中蔗果三糖、蔗果五糖、蔗果六糖、蔗果十糖4种低聚糖成分含量的差异具有统计学意义(P<0.05),其蔗糖、蔗果三糖、蔗果四糖、蔗果五糖、蔗果六糖、蔗果七糖、蔗果八糖、蔗果九糖的总量达16.47%。茅山苍术的果-果型低聚糖与蔗-果型低聚糖异构体的峰面积比值>1。结论:不同产地苍术中低聚糖的种类及含量差异较大,果-果型低聚糖与蔗-果型低聚糖峰面积比值>1可能是苍术道地性特征之一,可为该药材的深入开发利用及质量控制提供参考依据。

Objective:To establish a method for the analysis of oligosaccharides in Atractylodes lancea rhizome based on ultra-performance liquid chromatography tandem quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS)and a method for the quantification of oligosaccharides in A.lancea rhizome based on UPLC-evaporative light scattering detector(ELSD),and to investigate the oligosaccharide characteristics of A.lancea rhizome from different habitats.Method:UPLC-Q-TOF-MS was used to identify the oligosaccharides in A.lancea rhizome with the mobile phase of 0.1% ammonia acetonitrile solution(A)-0.1% ammonia solution(B)for gradient elution(0-0.5 min,98%A;0.5-2.0 min,98%-89%A;2.0-2.5 min,89%-86%A;2.5-5.5 min,86%-80%A;5.5-6.5 min,80%-72%A;6.5-9.5 min,72%-63%A;9.5-14.0 min,63%-50%A;14.0-16.0 min,50%A;16.0-16.5 min,50%-98%A;16.5-20 min,98%A),the column temperature of 60 ℃ and the flow rate of 0.2 mL·min-1.Electrospray ionization(ESI)was used to collect data in negative ion mode and the detection range was m/z 50-1 500.The qualitative analysis of oligosaccharides was accomplished by retention time,relative molecular weight,primary and secondary MS information of characteristic fragment ions in combination with reference substance information.UPLC-ELSD was employed to determine the contents of nine oligosaccharides in A.lancea rhizome with the mobile phase of 0.1% ammonia acetonitrile solution(A)-0.1%ammonia solution(B)for gradient elution(0-1 min,98%-75%A;1-7 min,75%-70%A;7-18 min,70%-55%A;18-23 min,55%A;23-23.5 min,55%-98%A;23.5-28 min,98%A),the drift tube temperature of ELSD was set at 50 ℃.Principal component analysis(PCA)and orthogonal partial least squares-discriminant analysis(OPLS-DA)were used to analyze the classification and differential components between A.lancea rhizome from different habitats.Result:A total of 24 oligosaccharides,containing 11 pairs of isomers,were identified from A.lancea rhizome.Among them,compared with samples from Anhui,Chongqing,Nanjing and Shaanxi,the contents of kestose(GF2),1F-fructofuranosylnystose(GF4),kestohexose(GF5),fructooligosaccharide DP10(GF9)in samples from Maoshan were statistically significant(P<0.05),and the total mass fraction of sucrose(GF1)-GF9 reached 16.47%.The peak area ratio of fructose-fructose oligosaccharide to its isomer sucrose-fructose oligosaccharide was greater than 1 in samples from Maoshan.Conclusion:The types and contents of oligosaccharides in A.lancea rhizome vary greatly among different habitats,and the peak area ratio of fructose-fructose oligosaccharide to sucrose-fructose oligosaccharide >1 may be one of the geoherb characteristics of A.lancea rhizome,which can provide a reference for the development,utilization and quality control of this herb.