【作者】 焦晶晶；

【导师】 张英；

【作者基本信息】 浙江大学 ， 食品科学， 2008， 博士

【副题名】单体制备、抗氧活性及其血管保护作用研究

【摘要】 本文以竹叶中特征性黄酮类化合物为研究对象,对四个竹叶碳苷黄酮的大鼠体内代谢行为进行了探索,得到了其吸收形式、吸收部位、代谢方式以及时相变化的信息,为竹叶抗氧化物(AOB)的人体安全性评价提供参数和依据。采用HPLC制备等技术,从AOB中分离制备两类特征性的黄酮单体,即四个碳苷黄酮(荭草苷、异荭草苷、牡荆苷、异牡荆苷)和一个多甲氧基黄酮(苜蓿素)。然后,以异荭草苷和苜蓿素为代表,选取木犀草苷、木犀草素和芹菜素为对照,采用Rancimat法、DPPH法、FRAP法以及化学发光法等六种抗氧化性能评价方法,探讨黄酮糖苷元与糖苷、碳苷与氧苷、甲基化和非甲基化的结构特征与其抗氧化和抗自由基活性之间的量效和构效关系。最后,采用兔离体主动脉环灌流模型,从高钾和苯肾上腺素(PE)预收缩两条途径研究竹叶黄酮对PDC和ROC的抑制作用,对KCl浓度-效应曲线及PE引起细胞内钙释放和外钙内流所引发血管收缩作用的影响,对高钾和鸟苷酸环化酶抑制剂亚甲基蓝预处理后的作用,对环氧合酶抑制剂吲哚美辛预处理后的作用,以及对一氧化氮合酶抑制剂L-NAME预处理后的作用；并用硝苯地平作为阳性对照药,研究竹叶黄酮的协同增效作用以及在细胞外有无钙离子存在的情况下对血管舒张因子NO释放量的影响,观察其对离体兔胸主动脉环的舒张血管效应,探讨可能的作用机制。主要研究结果归纳如下：(1)采用大鼠灌胃AOB的体内代谢模型,通过对主要脏器及消化道排泄物中目标化合物的分析检测,探讨碳苷黄酮的吸收形式、吸收部位、排泄方式以及随时相的变化,初步探明四种竹叶碳苷黄酮不经血尿途径吸收和代谢,而是经胃肠道途径进行代谢,与文献报道的氧苷黄酮的常规代谢方式存在明显差异。(2)以5升AOB浓缩液(固形物含量≥20%)为原料,通过柱层析(AB-8大孔吸附树脂)法得到6.5 g富含碳苷黄酮的馏分和174 g富含苜蓿素的馏分,然后采用制备型HPLC法分别从上述馏分中分离四种碳苷黄酮和苜蓿素,并用溶析结晶法和膜透析法纯化,分别得到荭草苷49 mg、异荭草苷142 mg、牡荆苷15 mg、异牡荆苷62 mg和苜蓿素3.09 g,纯度均大于90%。(3) Rancimat法测得在添加量为0.02-0.05%时,五种黄酮试样抗山茶油氧化的性能依次为：木犀草素>苜蓿素>木犀草苷>芹菜素>异荭草苷；DPPH法测得其清除DPPH的IC50为0.089-105.328mmol/L,活性强弱顺序为：木犀草素≈异荭草苷>木犀草苷>>苜蓿素>>芹菜素；FRAP法测得当添加量为0.02-0.1mmol/L时,五种试样的铁还原能力为：异荭草苷>木犀草素≈木犀草苷>苜蓿素>芹菜素；化学发光法研究表明,其清除0.2-的能力为苜蓿素>木犀草苷>异荭草苷>木犀草素>>芹菜素,IC50值范围为0.05-10.59mmol/L;清.OH能力为木犀草素>木犀草苷>苜蓿素>异荭草苷>>芹菜素,IC50值为0.24-691.03mmol/L;清H202能力为苜蓿素>木犀草苷>木犀草素>异荭草苷>>芹菜素,IC50值为0.37-130.58mmol/L。(4)采用兔离体主动脉环灌流模型,观察竹叶黄酮(总黄酮含量≥50%)对血管环的直接作用,发现竹叶黄酮对PE和高钾引起的预收缩血管环具有舒张作用,并呈浓度依赖关系。提示竹叶黄酮可能抑制了高钾和PE引起的外钙内流或者是抑制了细胞内两种不同钙池的钙释放,从而降低了细胞内钙离子的浓度而产生血管舒张作用。且此作用表现为非内皮依赖,表明竹叶黄酮是直接作用于血管平滑肌细胞而产生作用。对比人体脐带静脉内皮细胞株在胞外有无钙离子时NO的释放量,说明胞外钙离子只是增强了其产生NO的能力。加入一氧化氮合酶(NOS)抑制剂L-NAME和鸟苷酸环化酶(sGC)抑制剂亚甲基蓝后,竹叶黄酮的扩血管作用明显减弱,揭示其舒张血管的作用是通过NO介导途径,因此推论NO-sGC-cGMP途径可能介导了竹叶黄酮的舒张血管作用。竹叶黄酮还显示出与硝苯地平(阳性对照药物)的协同增效作用,用环氧合酶抑制剂吲哚美辛(10pmol/L)预处理,当竹叶黄酮试样浓度大于3000μg/mL时、对PE预处理的离体血管环具有显著的舒张效果(p<0.05,p<0.01),显示高浓度的竹叶黄酮舒张血管作用与环氧酶(通过产生前列腺素)有一定的关系。更多还原

【Abstract】 This study at first investigated in vivo the metabolic behavior of four flavone C-glycosides, characteristic flavonoids in bamboo leaves, when experimental rats took up the antioxidant of bamboo leaves （AOB）. Based on an effective HPLC inspective method, the research of metabolic kinetics of AOB was conducted. Some important information including absorption form and site, metabolic pathway and concentration change of objective compounds in different time phases were obtained, in order to supply much more scientific evidence on AOB for its certification of GRAS, generally regard as safe. Then, two kinds of characteristic flavonoids, i.e. four flavone C-glycosides （orientin, homoorientin, vitexin and isovitexin） and a multiple-methoxyl flavone （tricin） were isolated and obtained from AOB mainly using by the preparative HPLC method. Subsequently, the antioxidant activities of homoorientin and tricin were evaluated using by Rancimat, DPPH, FRAP and chemiluminescence methods, taking luteolin-7-O-glucoside, luteolin and apigenin as comparative compounds. The relationship between antioxidant capacity and chemical structure, such as aglycones and glycosides, C-glycosides and O-glycosides, methoxylation and non-methoxylation, were studied for elucidating the dose-dependent and/or structure-dependent relationships of flavonoids. Furthermore, the effect of bamboo-leaf-flavonoids （BLF） on the inhibition of potential dependent Ca²⁺channel （PDC） and receptor operated Ca²⁺channel （ROC） was studied via the hyperpotassium-induced and phenylephrine （PE）-induced pre-constriction pathways in a perfusion model of isolated thoracic aortic rings from rabbits. This study also investigated the effects of BLF on cumulative concentration-response curves with KC1 and PE-induced vasoconstriction from intracellular Ca²⁺ release and extracellular Ca²⁺influx; effects on the pre-treatment of the hyperpotassium and guanylate cyclase （sGC） inhibitor, i.e. methylene blue （MB）; effects on the pre-treatment of the cyclooxygenase inhibitor, i.e. indomethacin; and effects on the pre-treatment of the nitric oxide synthase （NOS） inhibitor, i.e. L-NAME. The present study further observed the synergistic effects of BLF on the NO production with and without the presence of extracellular Ca²⁺using the Ca²⁺ antagonist of nifedipine as a positive control, and demonstrated their vasodilatation effects on the thoracic aortic rings isolated from rabbits and relative mechanism. Results were summarized as follows:a) Based on our established HPLC method to effectively determine the contents of flavone C-glycosides in various biological samples, the absorption form and site, metabolic pathway and level change of objective compounds were obtained via the inspection of organs and excreta extracts after the oral administration of AOB in rats. It has been primary elucidated that four flavone C-glycosides were absorbed and metabolized via the gastrointestinal pathway, instead of the blood and urine pathway, which was significant different from the routine metabolism pathway of flavone O-glycosides.b) Totally 6.5 g of flavone C-glycoside rich fraction and 174 g of tricin-rich fraction was obtained from 5 L of AOB concentrated solution via the AB-8 macroporous adsorption resin based column chromatography. Then, flavone C-glycosides and tricin were isolated from the above fractions by the preparative HPLC method and further purified by the drowning-out crystallization and membrane dialysis methods. Totally 49 mg of orientin,142 mg of homoorientin,15 mg of vitexin,62 mg of isovitexin and 3.09 g of tricin were finally obtained all with a purity of more than 90%.c) The antioxidant activities of selected flavonoid samples evaluated by Rancimat method with the addition level range of 0.02-0.05%were ordered as follows:luteolin> tricin> luteolin-7-O-glucoside> apigenin> homoorientin. The DPPH’scavenging ability was ordered as follows:luteolin≈homoorientin> luteolin-7-O-glucoside >> tricin >> apigenin. Relative IC₅0 value range is 0.089-105.328 mmol/L. The ferric reducing ability with the addition level range of 0.02-0.1 mmol/L was ordered as follows:homoorientin> luteolin≈luteolin-7-O-glucoside> tricin> apigenin. The O₂ scavenging ability was ordered as follows:tricin> luteolin-7-O-glucoside> homoorientin> luteolin >> apigenin. Relative IC₅0 value range is 0.05-10.59 mmol/L. The’OH scavenging ability was ordered as follows:luteolin> luteolin-7-O-glucoside > tricin> homoorientin >> apigenin. Relative IC₅0 value range is 0.24-1691.03 mmol/L. The H₂O₂ scavenging ability was ordered as follows:tricin> luteolin-7-O-glucoside> luteolin> homoorientin >> apigenin. Relative IC₅0 value range is 0.37-130.58 mmol/L.d) The effect of BLF, which is a standard preparation with total flavonoid content above 50%, on the vascular rings was observed in a perfusion model of isolated thoracic aortic rings from rabbits. Results showed that there is a concentration-dependent relationship of the vasodilatation effect of BLF on PE-and hyperpotassium-induced pre-constricted vascular rings, which indicated that BLF may inhibit intracellular Ca²⁺release or extracellular Ca²⁺influx induced by hyperpotassium and PE, respectively. And so it presented a vasodilatation effect via reducing the intracellular Ca²⁺levels. Such effect was presented as the non-endothelium related form, which indicated that BLF exerted the effect directly via vascular smooth muscle cells. Comparison study of NO production with and without the presence-of extracellular Ca²⁺in the human umbilical vein endothelium cell line demonstrated that extracellular Ca²⁺only enhanced its NO production. The vasodilatation effect of BLF was significantly reduced after the addition of L-NAME and MB, which indicated their mechanism of action via the NO-sGC-cGMP pathway. Furthermore, BLF showed a synergistic effect with the presence of nifedipine, a positive control. After the pretreatment of indomethacin at 10μmol/L, a significant vasodilatation effect on PE pretreated isolated vascular rings was observed when the addition level of BLF was above 3000μg/mL （p<0.05,p<0.01）, and that indicated the vasodilatation effect of BLF in a high level may be related to cyclooxygenase via the production of prostaglandin.更多还原