摘要
苦荞麦是蓼科(Polygonaceae)荞麦属(Fagopyrum Mill)一年生草本植物,具有辅助降糖、抗氧化以及抗肿瘤等药理作用。近年来苦荞麦被加工成各种保健食品。目前,关于苦荞麦化学成分、代谢产物分析和鉴定的研究较少。本论文对苦荞麦的化学成分、分析方法及体内代谢产物三个方面进行了研究,为深入认识苦荞麦药效物质基础以及合理进行质量控制和资源开发利用奠定基础。
首先采用色谱分离技术,从苦荞麦70%乙醇提取物中提取分离得到11个主要化学成分,通过紫外光谱(UV)、质谱(MS)和核磁共振(NMR)技术鉴定化合物的结构。11个主要化学成分包括糖苷类、酰胺类和黄酮类化合物,分别是1,3,6'-tri-p-coumaroyl-6-feruloyl sucrose (1),3,6-di-p-coumaroyl-1,6'-di-feruloyl sucrose (2),1,6,6'-tri-feruloyl-3-p-coumaroyl sucrose (3),1,3,6,6'-tetra-feruloyl sucrose (4), N-trans-feruloyltyramine (5), Quercetin-3-O-β-D-galactoside (6), Quercetin-3-O-β-D-glucoside (7), Kaempferol-3-O-β-D-galactoside (8), Kaempferol-3-O-β-D-glucoside (9), Quercetin-3-O-α-L-rhamnoside (10), Quercetin-3-O-[β-D-xylosyl-(1→2)-α-L-rhamnoside](11),其中1个化合物(4)为新化合物,有4个化合物(2、3、5、11)首次从该属中分离得到。体外活性筛选发现糖苷类化合物(2、3、4)、酰胺类化合物(5)和部分黄酮类化合物(10和11)具有较好的抗氧化活性,糖苷类化合物(2和3)具有较好的肝保护活性。
为了进一步深入分析和鉴定苦荞麦中微量和难以分离得到的化学成分,采用HPLC-PDA/LTQ-FTICRMS技术,建立了苦荞麦70%乙醇提取物中化学成分的分析方法,采用Kinetex C18反相色谱柱(4.6×100mm,2.6μm, Phenomenex),以0.1%冰醋酸水溶液和乙腈为流动相,梯度洗脱,流速0.5mL/min;柱温35℃；进样量2μL；检测波长320nm;分流比1：1,采用ESI离子源正离子检测模式。一级质谱采用全扫描模式获得高分辨质谱数据,通过数据依赖性扫描模式获得多级质谱数据。分析了11个单体化合物的高分辨质谱数据、多级质谱数据和特征紫外吸收光谱等信息,总结了已知化学成分的质谱裂解规律；基于总结的质谱裂解规律和检测到苦荞麦中其他化合物的高分辨质谱数据和多级质谱数据以及紫外光谱等信息,从苦荞麦提取物中共计鉴定了36个化学成分,其中15个化学成分确定其结构,另外21个化学成分通过质谱数据推测了它们可能的结构。
为了能够同时定量分析苦荞麦中极性差别大、结构类似的多种化学成分,采用在线中心切割二维高效液相色谱技术,建立了苦荞麦中12个化学成分定量分析方法。第一维采用Acclaim Mixed-Mode HILIC-10(2.1×10mm,3μm)色谱柱,第二维采用Acclaim RSLC Phenyl-1(2.1×150mm,3μm)和Acclaim RSLC PolarAdvantage Ⅱ (2.1×150mm,2.2μm)两根色谱柱,通过两个切换阀以并联的方式连接；柱温40℃；检测波长320nm;流速0.5mL/min;进样量4μL；以0.03%磷酸水溶液和乙腈为流动相,洗脱方式为梯度洗脱。利用阀切换通过正向和反向冲洗相结合的洗脱方式,将苦荞麦提取物分成两个部分,一部分主要含有黄酮和酰胺类化合物,另一部主要含有糖昔类化合物,两个部分分别在不同固定相的第二维色谱柱中进行分离分析,结果使得极性相差较大、结构类似的12个化合物实现完全分离。方法验证结果显示,所建立的在线中心切割二维高效液相色谱分析方法能够满足苦荞麦中12个化学成分准确测定的要求,重复性(RSD<3.4%),日内精密度和日间精密度(RSD<4.6%),加样回收率(91.21-107.76%),检出限(0.05-0.21μg/mL)和定量限(0.10-0.41μg/mL)。表明所建立的方法准确、可靠,能够对苦荞麦不同部位以及全草进行定量分析,为评价苦荞麦的质量和资源合理开发,提供可靠的数据支持。在线中心切割二维高效液相色谱分析方法与常规HPLC分析方法相比,显著提高了分离的选择性,能够同时定量分析中草药中的不同结构类型以及同类型结构相似的多个化学成分。
大鼠原位肝肠灌流模型能够有效模拟大鼠体内肝肠代谢情况,具有基质相对简单,原药用量少等优势。因此采用大鼠原位肝肠灌流模型对从苦荞麦中分离得到的有效成分酰胺类、黄酮类和糖苷类三类11个化学成分进行代谢产物研究。建立了HPLC-PDA/LTQ-FTICRMS分析方法,分析灌流液、肠内容物以及胆汁样品中的代谢产物。采用Kinetex C18反相色谱柱(4.6×100mm,2.6μm, Phenomenex)色谱柱；流动相采用0.1%冰醋酸水溶液和乙腈体系,梯度洗脱,进样量2μL；检测波长320nm;柱温35℃,流速0.6mL/min。选用ESI离子源,采用正离子检测模式,一级质谱采用全扫描模式获得高分辨质谱数据,采用数据依赖性扫描模式获得多级质谱数据。通过对灌流液、肠内容物和胆汁样品中检测到代谢产物的高分辨质谱质谱数据、多级质谱数据和紫外吸收光谱等信息进行分析,结果发现酰胺类化合物的7个代谢产物,黄酮类化合物的17个代谢产物,糖苷类化合物的10个代谢产物,共计发现34个代谢产物。糖苷类化合物的代谢转化反应主要包括酯水解的Ⅰ相代谢,酰胺类和黄酮类化合物的代谢反应主要为甲基化、硫酸化和葡萄糖醛酸化的Ⅱ相代谢。
基于11个单体化合物在大鼠原位肝肠灌流模型上代谢产物的研究结果,进一步探索了苦荞麦提取物复杂体系在大鼠体内的代谢情况。采用大鼠经口灌胃苦荞麦提取物方式,分别收集给药后0-24h和24-48h的粪便和尿液样品；0-12h、12-24h和24-48h的胆汁样品；以及0.5h、1h、3h和6h的血浆样品。样品前处理后,采用建立的大鼠肝肠灌流模型样品的HPLC-PDA/LTQ-FTICRMS分析方法。在粪便中发现和鉴定了5个原型成分(P2,3,4,5,6)和3个代谢产物；在尿液中发现和鉴定了1个原型成分(P3)和代谢产物6个；在胆汁样品中发现和鉴定了1个原型成分(P1)和代谢产物10个；在血浆样品中发现代谢产物3个。共计分析和鉴定了23个化合物,其中17个代谢产物,6个原型成分。苦荞麦提取物在大鼠体内的代谢转化反应与各个单体化合物在大鼠原位肝肠灌流模型上的代谢反应类似,主要为酯水解的Ⅰ相代谢、葡萄糖醛酸化和硫酸化的Ⅱ相代谢,结合已有的研究结果阐述了苦荞麦化学成分在大鼠体内的代谢特点和代谢途径。
Tartary buckwheat belongs to the Polygonaceae family. Tartary buckwheat (Fagopyrum tataricum Gaertn) has antidiabetic, antioxidant and antirumor activities. In recent years, tartary buckwheat has been used to make various healthful foods. However, its chemical constituents and metabolites in vivo have not yet been fully characterized and identified. In this thesis the chemical composition, analytical method and metabolism in vivo of tartary buckwheat were studied in order to elucidate the active substances and provide scientific evidences for quality control and rational use.
Eleven compounds had been isolated from70%ethanol extract of tartary buckwheat by various chromatographic techniques. These structures were elucidated by UV, MS and NMR experiments, including phenlypropanoid glycosides, nitrogen compound and flavonoids, namely1,3,6-tri-p-coumaroyl-6'-feruloyl sucrose (1),3,6-di-p-coumaroyl-1,6'-di-feruloyl sucrose (2),1,6,6'-tri-feruloyl-3-p-coumaroyl sucrose (3),1,3,6,6'-tetra-feruloyl sucrose (4), N-?rans-feruloyltyramine (5), quercetin-3-O-β-D-galactoside (6), quercetin-3-O-β-D-glucoside (7), kaempferol-3-O-β-D-galactoside (8), kaempferol-3-O-β-D-glucoside (9), quercetin-3-O-a-L-rhamnoside (10), quercetin-3-O-β-D-xylosyl-(1→2)-a-L-rhamnoside](11).1,3,6,6'-tetra-feruloyl sucrose (4) named taroside as a new phenlypropanoid glycoside, together with3,6-di-p-coumaroyl-1,6'-di-feruloyl sucrose (2),1,6,6'-tri-feruloyl-3-p-coumaroyl sucrose (3), N-trans-feruloyltyramine (5) and quercetin-3-O-[β-D-xylosyl-(1→2)α-L-rhamnoside](11) were isolated for the first time from the Fagopyrum species. The partial phenlypropanoid glycosides (2,3and4), nitrogen compound (5) and flavonoids (5and11) have good antioxidant and phenlypropanoid glycosides (2and3) have good liver protective activities in vitro.
In order to comprehensively identify the minor and hardly separated chemical compositions in tartary buckwheat, a novel high performance liquid chromatography coupled with photodiode array detector and linear ion trap FTICR hybrid mass spectrometry (HPLC-PDA/LTQ-FTICRMS) method was established. HPLC separation was performed on a Kinetex C18reverse phase column (4.6x100mm,2.6μm, Phenomenex) by gradient elution using water containing0.1%acetic acid and acetonitrile as mobile phase at a flow rate of0.5mL/min. The eluent from HPLC system was split (1:1) and sprayed into mass spectrometer with an electrospray interface operating in positive ionization. The high resolution and accurate mass spectral data were obtained by the first stage full-scan FTICRMS analysis. MS" data were acquired by data-dependent mode of multiple stage mass analysis. The retention time, maximum UV absorption wavelength, accurate mass weight and characteristic fragment ions of11single compounds were collected on line and their characteristic fragment pattern were summaried. On the basis of the retention time, UV spectral data, accurate molecular weight and mass fragmentation pattern of detected compounds in tartary buckwheat, a total of36chemical compositions that15compounds were unequivocally identified and21compounds tentatively assigned.
An on-line heart-cutting two-dimensional high performance liquid chromatography (2D-LC) coupled with a diode array detector was explored to determine the different polarities and similarly structural chemical compositions in tartary buckwheat. The combination of various stationary phases were developed and optimized with two switch valves as the interface. Acclaim Mixed-Mode HILIC-10(2.1x10mm,3μm) was used for the first dimensional column. Acclaim RSLC Phenyl-1(2.1x150mm,3μm) and Acclaim RSLC Polar Advantage Ⅱ (2.1x150mm,2.2μm) in parallel were used for the second dimensional columns. The mobile phase was acetonitrile and0.03%aqueous phosphoric acid in water. The temperature in the compartment of the autosampler was20℃, the column temperature was40℃, the injection volume was4μL, and UV absorbance was detected at a wavelength of320nm. The on-line heart-cutting automated2D-LC system was successfully developed by means of valve switching and back-flush. The optimum2D-LC system was established and initially validated to profile and simultaneously determine12major constituents in tartary buckwheat. It showed good performance in terms of repeatability (R.S.D.<3.4%), intra-day and inter-day precision (R.S.D.<4.6%), recovery (91.21-107.76%), limit of detection (0.05-0.21μg/mL), and limit of quantification (0.10-0.41μg/mL). The established2D-LC method was accurate and reliable to determine12major compounds in the different parts and entire plant of tartary buckwheat and provide the reference for quality control and resonable use of tartary buckwheat.
The in situ liver-intestinal perfusion model can simulate enterohepatic metabolism. In addition, its advantages are beneficial for metabolites identification due to the relatively simple biological matrix and the small amount of drug. Eleven single compounds of three chemical classes including nitrogen compound, flavonoids and phenlypropanoid glycosides were investgated on this model. A HPLC-PDA/LTQ-FTICRMS method was established. Chromatographic separation was carried out on a Kinetex C18reverse phase column (4.6×100mm,2.6μm, Phenomenex). The mobile phase was acetonitrile and0.1%acetic acid aqueous solution in water. The flow rate was set at0.6mL/min, and the column temperature was maintained at35℃. The injection volume was2μL. The positive ESI mode was performed. The high resolution and accurate mass spectral data were obtained by the first stage full-scan FTICRMS analysis. MS" data were acquired by data-dependent mode of multiple stage mass analysis. On the basis of the retention time, UV spectral data, accurate molecular weight and mass fragmentation pattern of detected compounds in biological samples. A total of thirty-four metabolites, that is, seven metabolites from nitrogen compound, seventeen metabolites from flavonoids, ten metabolites from phenlypropanoid glycosides were found and identified. Metabolic reaction include the phase I metabolism of ester hydrolysis and the phase Ⅱ metabolism of methylation, sulfation and glucuronidation.
On the basis of metabolism of eleven single compounds on the in situ liver-intestinal perfusion model, the metabolites of tartary buckwheat extract in rats were probed. Fece and urine samples during0-24h and24-48h, bile samples during0-12h,12-24h and24-48h and plasma samples at0.5h,1h,3h, and6h were collected after oral administration of the tartary buckwheat extract to rats. After the pretreatment of biological samples, the established HPLC-PDA/LTQ-FTICRMS method for liver-intestinal perfusion study was applied to analyze metabolites. In feces, three metabolites derived from hydrolysis of the phenlypropanoid glycosides and five phenlypropanoid glycosides (P2,3,4,5and6) were detected and identified. Six metabolites including N-trans-feruloyltyramine glucuronidate and sulfate as well as kaempferol glycosides glucuronidation and one prototype drug (P3) were found and identified in urine. In addition, ten metabolites and one prototype drug (P1) were identified in bile. Three metabolites including quercetin-diglucuronide, N-/rans-feruloyltyramine glucuronide and kaempferol-monoglucuronide were identified in plasma. A total of twenty-three compounds including seventeen metabolites as well as six prototype drugs were identified. The metabolic pathways of tartary buckwheat extract in rats were similar with those of eleven single compounds on the in situ liver-intestinal perfusion model. The phenlypropanoid glycosides were easily underwent by the phase Ⅰ metabolic reaction of ester hydrolysis and the nitrogen compound and flavonoids were mainly metabolized by phase Ⅱ metabolic reaction of methylation, sulfation and glucuronidation. Moreover, the metabolic pathways of nitrogen compound, flavonoids and phenlypropanoid glycosides were proposed.
引文
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