摘要
本论文开展了以黄酮醇类化合物为主要成分，具有抗老年痴呆和抗血栓活性的草棉植物活性提取物AB-8-2在大鼠体内代谢转化的系统研究。提出了以质谱裂解行为与代谢途径特征相结合获取双重信息且互补参照的研究思路，并将原型成分和代谢产物的结构推断互相佐证，建立了活性提取物AB-8-2中黄酮醇类化合物在大鼠体内生物转化的整体代谢轮廓质谱分析方法。同时还比较研究了黄酮醇类单体成分血浆中代谢产物的特征以及黄酮醇苷类化合物在不同CID方式下质谱裂解行为的差异。
主要包括以下4个方面的内容：
1．黄酮醇单糖苷类化合物在不同CID方式下的裂解行为比较
选择有代表性的几种黄酮醇苷类单体化合物，进行了在ESI-MS~2裂解行为的比较研究，探讨了3D离子阱、Q TRAP~(TM)型质谱仪的EPI、LIT-MS~2谱扫描的三种不同CID方式下糖基化位置对该类化合物裂解行为的影响及其特征性裂解反应，研究发现不同CID方式将导致以糖基裂解方式为主的特征性差异，糖基丢失产生的苷元离子[Y_0-H]~(-·)和苷元离子Y_0~-相对强度比值及另一个诊断离子~(0，2)X_0~-产生与否与CID方式密切相关。不同类型质谱仪上获得的化合物结构判断规律不能不加以验证地应用于在其它类型质谱仪上采集的数据。但是，不同原理及类型的MS／MS仪获得的结果可以提供很有意义的参考价值及补充性的重要信息。
2．草棉植物活性提取物中黄酮醇类成分及其整体代谢轮廓的质谱分析方法研究
在前期研究的基础上，采用高效液相色谱-多级质谱联用技术(HPLC-MS~n)，开展了对AB-8-2及其经灌胃和静注给药后大鼠胆汁样品的系统分析研究。首先从AB-8-2(原型)中分析共检测到31个黄酮醇类化合物；包括一些极微量且在紫外色谱上没有吸收峰的黄酮醇双糖苷和乙酰基糖苷类化合物，并根据MS~n谱对此进行细致分析，推断了它们的结构。本研究结合原型组分的质谱裂解规律和黄酮醇类成分的代谢途径特征，以不同给药途径获得的大鼠胆汁样品为研究对象，建立了黄酮醇类混合物及其代谢整体轮廓分析的快速质谱分析方法。运用本研究方法在AB-8-2及大鼠胆汁样品中共分析鉴别出了58个以槲皮素或山柰酚为母核的硫酸化、甲基化、葡萄糖醛酸化Ⅱ相代谢产物及糖苷原型，其中包括多组同分异构体。本项研究中提出的黄酮醇类化合物原型及其在生物体液中代谢产物多组分的快速、整体轮廓分析的方法，为中药及天然药物的复杂活性组分体系的代谢研究提供了一种新的研究思路。
3．大鼠灌胃给予活性提取物AB-8-2后尿液中的代谢物分析
研究了大鼠口服给予AB-8-2后尿液中的代谢转化情况，在一个分析周期内检测出了灌胃给予AB-8-2后大鼠尿样中的黄酮醇类成分。共鉴定了51个以槲皮素或山柰酚为母核的Ⅱ相代谢产物及黄酮醇苷元，发现在尿样中以槲皮素双葡萄糖醛酸结合物存在的代谢产物为主要形式。同时与口服给予AB-8-2后胆汁样品的结果比较分析，发现两种生物样本的代谢产物种类存在很大差异。其中尿样中发现的黄酮醇代谢终产物主要是葡萄糖醛酸结合物，且以甲基化槲皮素的单葡萄糖醛酸结合物含量最高，但是没有观察到像胆汁中多样化的代谢反应产物，没有检测到乙酰化、硫酸化产物。此外，还发现AB-8-2中某些黄酮醇双糖苷类化合物在小肠吸收过程中可保留其中一个糖基，并以次级苷的形式与葡萄糖醛酸结合，随尿液被排出体外。尿液样品中一些组分含有共流出的葡萄糖醛酸甲酯结合物，对此，经分析判断为在样品处理过程中产生的人工代谢产物。
4．静脉注射槲皮素及其不同糖苷类化合物后大鼠血浆中的代谢物比较分析
对比分析了大鼠静脉给予槲皮素及其C-7、C-3′、C-3位糖苷取代的4种黄酮醇类化合物后大鼠血浆中的代谢产物，经研究发现内源性物质在槲皮素母核上加合位点的倾向性；同时表明C-7位的游离羟基对葡萄糖醛酸结合反应具有重要影响。此外，还分析了血浆中检测不到4′-OH甲基化代谢产物的原因，提出是由于3′-OH被葡萄糖取代后，B环的邻二酚羟基结构消失，导致槲皮素-3′-O-葡萄糖苷不能作为儿茶酚甲基转移酶(COMT)的底物，因此不发生甲基化代谢反应。进一步阐明了对黄酮醇类化合物而言，只有母核上具有儿茶酚结构在体内方可发生甲基化反应。
AB-8-2 was an active fraction obtained from Gossypium herbaceam L, which mainly consisted of flavonol glycosides. The metabolism biotransformation of AB-8-2 in rat was investigated systematically using high-performance liquid chromatography/multi-stage tandem mass spectrometry (HPLC-MS~n). A novel strategy for the rapid identification of flavonols in complex herbal extract and their metabolic pathway was proposed and employed in profiling the integral metabolism of flavonols of AB-8-2.
The characteristic mass spectrometric fragmentation behavior of flavonol O-glycosides were investigated in detail by ESI-MS/MS technique in negative ion mode using three CID mode on two types of mass spectrometer. The metabolites of flavonols in the bile and urine samples after administistration of AB-8-2 were analyzed using HPLC-MS~n in a single chromatographic run. In addition, metabolites in plasma of rats after a single intravenous dose of quercetin and its three glycosides, respectively, were characterized and compared for the purpose of investigating the region-selectivity of conjugation of quercetin.
1. Characteristic fragmentation behavior of flavonol mono-glycosides with different CID mode
The mass spectrometric fragmentation behavior of several typical flavonol O-glycosides were investigated in detail by ESI-MS/MS technique in negative ion mode using three CID mode on two types of mass spectrometer. The typical MS~2 spectrum obtained on a 3D ion trap, enhanced product ion (EPI) spectrum and "trap-like" MS~2 spectrum obtained on Q TRAP~(TM) mass spectrometer were compared. The fragmentation behavior of flavonol O-glycosides under different CID conditions were not identical but can provide complementary information revealing the structural differences in glycosylation position. In negative ion mode, the glycosylation position of flavonol 3-O-glycosides, flavonol 7-O-glycosides and flavonol 3'-O-glycosides were differentiated and determined through investigating the fragmentation behavior of [M-H]~- ions resulting from the different glycosylation position. [Y_0-H]~(-·) and ~(0,2)X_0~- ion were used as diagnostic ions in differentiating 3-O, 7-O and 3'-O-glycosyl flavonol using 3D ion trap, while the relative abundance of [Y_0-H]~(-·) ion can be employed to characterize the three isomers without the accessorial information of ~(0,2)X_0~- ion using EPI spectrum. The results provided a basis for structure identification using fragmentation rule derived from different instrument.
2. Fast profiling of the integral metabolism of flavonols in the active fraction of Gossypium herbaceam L. using HPLC-MS~n technique
The intergral metabolism of flavonols of AB-8-2 was profiled based on the combination of fragmentation behavior and metabolic pathways. Fifty-eight flavonols as a range of mixed sulphate, methyl, glucuronide and glycoside derivatives of quercetin or kaempferol were detected in rat bile samples, including several groups of isomers. The metabolic differences in bile samples from rats after oral and intravenous administration were compared to evaluate the influence of intestinal metabolism. The relationships between metabolites and parents were elucidated for some components. By profiling the constituents in AB-8-2 and metabolites in bile, a integral view on the biotransformation of the constituents in AB-8-2 was obtained.
3. Metabolites of flavonols in urine after oral administration of AB-8-2
Based on the method established, the urine sample after oral administration of AB-8-2 was analyzed. In one analytical run, 51 constituents including 35 di-glucuronidated metabolites, 12 mono-glucuronidated metabolites and 4 flavonol aglycones were characterized. Flavonol diglycosides in AB-8-2 probably remained one glycoside in the structure during the absorption process in intestinal tract. Some co-eluated glucuronide methyl ester conjugated metabolites were also identified in the urine sample, which may be produced in the process of sample preparation.
4. Comparsion of the metabolites in rat plasma after intravenous administration of quercetin and its three glycosides
By employing the HPLC-MS and HPLC-MS/MS method, the metabolites in the rat plasma were characterized after intravenous administration of quercetin, quercimeritrin, quercetin-3'-O-glucoside and hyperoside, respectively. The results indicated that quercetin occupied the most various metabolic reactions among these four compounds investigated. No glucuronidated conjugates was found in the plasma sample after intravenous administration of quercimeritrin, which suggested that the free 7-OH was an essential group for the glucuronidation. For quercetin-3'-O-glucoside, no methylation metabolite was detected in the plasma sample, which futher confirmed that catechol structure in B ring was necessary for the methylation in vivo for flavonols. As for hyperoside (quercetin-3-O-galactose), two metabolites were identidied as hyperoside monoglucuronide and methylated hyperoside, respectively.
引文
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[1] Shahat AA, Cuyckens F, Wang W, Abdel-Shafeek KA, Husseiny HA, Apers S, Van Miert S, Pieters L, Vlietinck AJ, Claeys M. Structural characterization of flavonol di-O-glycosides from Farsetia aegyptia by electrospray ionization and collision-induced dissociation mass spectrometry. Rapid Commun. Mass Spectrom. 2005; 19: 2172-2178.
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[4] Ablajan K, Abliz Z, Shang XY, He JM, Zhang RP, Shi JG. Structural characterization of flavonol 3, 7-di-O-glycosides and determination of the glycosylation position by using negative ion electrospray ionization tandem mass spectrometry. J. Mass Spectrom. 2006; 41: 352-360.
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