木瓜复合抗氧化体系的重组及其抗ApoE基因敲除小鼠动脉粥样硬化的生物学效应研究
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摘要
近年来关于动脉粥样硬化(AS)发生机制研究较多的是“损伤应答学说和炎症免疫学说”,其认为动脉粥样硬化疾病表现了一种高氧化应激状态,它以血管壁中脂质和蛋白质氧化为特征,活性氧(ROS)与炎性因子相互作用,贯穿AS的发生发展过程。抗氧化剂及抗炎症药物用于AS及心血管疾病的防治效果已获得很多实验室及临床研究证实。天然植物中含有的抗氧化物质,通常也伴随有抗炎症的效应,例如黄酮、原花青素等多酚类物质已被很多研究证明在AS防治过程中,一方面是提高了机体抗氧化状态,减少ROS导致的LDL氧化程度;另一方面是下调一些炎性因子表达及抑制巨噬细胞源泡沫细胞中脂质聚积、减轻炎症反应,从而起到较好的抗动脉粥样硬化作用(Antiatherogenic effect)。木瓜含有丰富的抗氧化物质,如维生素C、多酚类物质及SOD活性类物质,本研究小组在辐照模型小鼠实验中证实木瓜水相提取物具有升高血清超氧化物歧化酶(SOD),减少脂质过氧化物丙二醛(MDA)的作用;在Ⅱ型胶原诱导关节炎大鼠模型中,木瓜苷干预模型鼠后,通过抑制局部炎症介质的产生,缓解了关节炎症状;从木瓜提取的萜类物质还被证实有抑制与AS病变有关的组织因子(TF,又名组织凝血致活酶)活性的作用。
目前对于通过抗氧化效应防治AS的研究主要关注的抗氧化物类别是维生素类、植物化合物及人工合成的抗氧化剂,多见于维生素E、维生素C、植物黄酮、原花青素和普罗布考等。将其中具有阳性结果的研究所涉及到的防治AS机制进行总结,有如下几个方面:(1)提高了血清抗氧化还原能力(T-AOC);(2)降低了血清MDA的水平;(3)抑制了LDL的氧化;(4)降低了泡沫样细胞的形成;(5)上调了GSH-Px、SOD、过氧化氢酶的基因表达;(6)下调了巨噬细胞的聚集程度;(7)抑制了TNF-α基因的表达。这些抗氧化剂最终表现出来的抗AS组织病理学变化是中内膜厚度(IMT)最大值减小和斑块尺寸缩小。但不少的单一抗氧化剂干预AS实验并没有达到预期的结果,有越来越多的研究者认为抗氧化物之间的协同效应发挥非常重要,而以植物粗提物或某一大类复合抗氧化组分来研究抗AS作用所获得的实验结果越来越振奋人心。
因此,本研究选用含丰富抗氧化组分的木瓜(Chaenomeles speciosa)为原料,采用生物酶解、环糊精包合和喷雾制粒技术,将存在于植物体组织中的抗氧化物质抽提出来,对其进行包合修饰,重新将其固化在环糊精给药载体上,将这种重新组合(下简称:重组)的木瓜复合抗氧化物添加入高脂饲料中,喂饲ApoE基因缺陷(ApoE-/-)小鼠,对木瓜抗氧化体系防治AS的生物学效应及机制进行研究和探讨。
方法:
采用纤维素-果胶酶处理木瓜,通过正交实验设计获取最优酶解工艺;对提取的木瓜抗氧化复合物中的多酚类物质进行TLC薄层色谱鉴定,采用λ-环糊精包合技术对酶解提取的木瓜复合抗氧化物进行包合修饰;采用流化床喷雾制粒技术制备由β-环糊精为主要载体的重组木瓜复合抗氧化物颗粒(CSP);比较木瓜果实及CSP中维生素C含量、总酚量、单宁量以及FRAP、SOD的活性;采用改良的DPPH微孔板定量法(MQ)评测加工重组前后总抗氧化能力(TAC)的变化,并结合动力学曲线分析木瓜抗氧化体系重组前后其抗氧化特征变化。选用ApoE-/-雄性2月龄小鼠40只,随机分为5组,分别为空白对照组(Control)、CSP低剂量组(5%CSP)、CSP高剂量组(10%CSP)、超微茶粉组(Tea)、维生素C组(VC),在SPF级动物房喂养16周,眼眶取血测定血脂(TC、TG、LDL-C、HDL-C)、血清总抗氧化能力(T-AOC)和血清谷胱甘肽过氧化物酶(GSH-Px);免疫组化测定斑块TNF-α表达水平,免疫荧光检测CD68表达水平和巨噬细胞计数;HE染色分析主动脉窦斑块面积率及主动脉弓中内膜厚度(IMT);油红O染色降主动脉分析脂质沉积血管内膜状况,结合电镜扫描阐述动脉血管内壁微观变化。
主要结果和结论如下:
1.酶解处理木瓜果实体显著提高了木瓜出汁率,其最优酶解工艺为果胶酶添加量2%,纤维素酶添加量2%,温度40℃,时间60分钟。酶解技术的运用使木瓜主要抗氧化物——维生素C、总酚、单宁和黄酮提取总量分别提高了35.1%、43.8%、45.3%和46.7%。通过TLC色谱鉴定,木瓜提取物中含有儿茶素、表儿茶素和槲皮素、芦丁、绿原酸和原花青素。酶解使木瓜组织细胞壁崩解,这有利于完整地将木瓜原有的抗氧化组分浸提出来、重组到环糊精给药载体上。
2.γ-环糊精通过单一物质包合反应验证了其对木瓜含有的维生素C(呋喃烯醇式内酯结构)、原花青素(单宁类多聚体结构)、绿原酸(缩酚酸结构)和芦丁(类黄酮单元结构)具有包合作用。多酚类物质的包合常数在2.8×103～4.2×104之间,其Ka随多酚物质分子量增加而增大;γ-环糊精对维生素C的包合常数也达到了5.7×103。木瓜酶解提取复合物与γ-环糊精反应达到平衡时的包合常数为1.9×103,表明包合反应充分。包合修饰的实现有助于后期重组固化制粒的加工稳定性、耐储藏性及生物利用率的提高。
3.采用顶喷流化床喷雾制粒工艺,流化床体中心温度45℃、风机频率4~5kHz、进料泵3~5Hz、抖料器频率35~40次/min;获得的重组木瓜复合抗氧化物(CSP)每克含有维生素C、总酚、单宁含量分别为2.05±0.09mg、22.70±1.57mg和9.64±0.65mg,比重组前的木瓜抗氧化体系单位质量含有的维生素C、总酚和单宁量提高了219.8%、344.2%、348.4%;每克CSP的SOD活性、FRAP和TAC分别为700±21U、172.9±6.7μmol Fe2+、945±20μg DPPH?,比重组前的木瓜抗氧化体系单位质量的SOD活性、FRAP和TAC提高了236.5%、213.8%、300.4%。重组的木瓜抗氧化体系不仅提高了抗氧化物的含量,还比木瓜果实中抗氧化体系在储藏过程中更稳定,更易于在后期动物干预过程中保持应有的抗氧化活性。
4.改良创新了DPPH?微孔板测定技术(MQ)测定植物总抗氧化能力(TAC),不仅具有批处理量大、节约试剂和减少系统误差的优点,还能同时完成对抗氧化组分物质清除自由基反应动力学特征的测定。通过MQ评测木瓜抗氧化体系重组加工前后TAC及动力学曲线特征,推断重组后的木瓜抗氧化体系没有改变原有的抗氧化特性。
5.木瓜复合抗氧化物(CSP)在抗ApoE-/-小鼠动脉粥样硬化的生物学效应方面,其组织病理学变化主要表现为:CSP显著减少了降主动脉管壁内表面的脂质沉积发生,5%和10%的CSP干预后ApoE-/-小鼠降主动脉管壁内表面的脂质沉积面积率(OSL)分别为0.22和0.12;对比Control组,5%、10%的CSP添加分别使ApoE-/-小鼠降主动脉管壁内表面脂质沉积减少了40.8%和67.3%。CSP显著抑制了主动脉弓中内膜增厚的病变,低剂量和高剂量CSP干预后的ApoE-/-小鼠的主动脉弓中内膜厚度均值(IMT)分别为128.9和95.5;对比Control组,5%、10%的CSP添加分别减少了ApoE-/-小鼠主动脉弓中内膜30.1%和48.2%的增厚量。CSP显著减小了主动脉窦斑块的尺寸,5%和10%的CSP干预后ApoE-/-小鼠的主动脉窦斑块面积率分别为0.339和0.276;对比Control组,添加5%的CSP使ApoE-/-小鼠的主动脉窦斑块缩小了23.1%,添加10%的CSP使ApoE-/-小鼠的主动脉窦斑块缩小了38.1%。实验表明重组的木瓜抗氧化物从AS的发生阶段——“脂质沉积”到AS的发展阶段——“斑块形成”,都表现出了抗AS的生物学效应。
6.木瓜复合抗氧化物(CSP)抗AS的相关机制实验研究结果及结论:
1)木瓜复合抗氧化物显著降低了ApoE-/-小鼠的TC、TG、LDL-C水平。对比Control组血脂指标水平,添加5%的CSP分别使ApoE-/-小鼠血清TC和LDL-C降低了14%和21.2%;添加10%的CSP分别使ApoE-/-小鼠血清TC和LDL-C降低了17.3%和46.6%,而且还观察到了TG有36.6%的下降。实验结果充分表明重组的木瓜复合抗氧化物具有降低诱发AS相关血脂指标水平的作用,特别是降低主要风险因素——LDL-C的血清浓度。
2)木瓜复合抗氧化物显著提高了血清抗氧化的能力。对比Control组的血清抗氧化指标,添加5%的CSP分别使ApoE-/-小鼠血清GSH-PX和T-AOC提高了30%和53.3%;添加10%的CSP分别使ApoE-/-小鼠血清GSH-PX和T-AOC提高了47.1%和126.7%。实验结果表明,重组的复合木瓜抗氧化物不仅在体外FRAP、SOD和TAC抗氧化指标上表现出很强的抗氧化特性,还在体内发挥出了提高血清抗氧化能力的生物学抗氧化效应,这对AS发生发展的关键风险因子——ox-LDL的生成有潜在地抑制作用。
3)木瓜复合抗氧化物显著下调了ApoE-/-小鼠主动脉窦斑块中TNF-α的表达。以Control组主动脉斑块中TNF-α评分结果为基准,添加5%、10%的CSP使ApoE-/-小鼠主动脉窦TNF-α分值分别降低了19%和67%。实验结果表明重组的木瓜复合抗氧化物具有抗炎生物学效应,这对由炎性因子介导的AS发生发展效应有潜在的抑制作用。
4)木瓜复合抗氧化物显著下调了ApoE-/-小鼠主动脉窦部CD68的表达水平和减少了巨噬细胞数。以Control组免疫荧光结果为基准,添加5%、10%的CSP分别使ApoE-/-小鼠主动脉窦部巨噬细胞标记物CD68表达水平下调了25.1%和38.3%;添加5%、10%的CSP分别使ApoE-/-小鼠主动脉窦部巨噬细胞数减少了42.8%和50.3%。实验结果表明,重组的木瓜复合抗氧化物对巨噬细胞介导的AS促发效应有潜在的抑制作用。
5)通过电镜扫描观察,ApoE-/-小鼠实验模型有典型的AS发生发展表征,如脂质沉积引发了血管壁表面微观结构的变化,表现为内膜纹理走向紊乱、表面有“虫蚀”样、间隙增大,血管壁向管腔隆起增厚和血小板凝集变形沉积在管壁上。这些都与本实验探究的血脂异常、炎性因子水平、巨噬细胞数和机体抗氧化状态有密切联系和相关性。
7.重组的木瓜复合抗氧化物比发挥相似抗AS效应的茶粉添加来说,对ApoE-/-小鼠生长影响更小,干预过程更安全;重组的木瓜复合抗氧化颗粒比单一维生素C添加对ApoE-/-小鼠的AS防治更有效。
综上所述,本研究通过酶解提取、包合修饰和喷雾制粒技术将木瓜中的抗氧化组分成功地、较完整地重组到了环糊精给药载体上,制备成了具有良好储藏特性的木瓜复合抗氧化颗粒,其含有丰富的维生素C、多酚类物质,具有清除超氧阴离子、脂质自由基和铁离子还原能力;通过ApoE-/-小鼠动脉粥样硬化干预实验模型证实了重组的木瓜复合抗氧化物具有防治动脉粥样硬化的功效,主要体现为减少降主动脉内壁脂质沉积、抑制主动脉弓中内膜厚度的增加、抑制主动脉窦斑块的增长。其可能的机制是木瓜抗氧化物通过降低血清TG、TC和LDL-C的浓度水平、提高机体抗氧化能力(血清GSH-PX和T-AOC的增加)从而抑制LDL的氧化程度、降低炎性因子的表达及炎性细胞的数量发挥抗动脉粥样硬化的生物学效应。实验还通过对比研究添加超微绿茶粉(多酚类抗氧化物为主的抗氧化剂)和维生素C对ApoE-/-小鼠动脉粥样硬化的防治功效,证实了含有总酚量相近的超微绿茶粉在很多生物学效应上与添加木瓜复合抗氧化物的低剂量组一致,从一个侧面证实了木瓜复合抗氧化物中的多酚类物质与目前研究公认的防心血管疾病茶多酚类物质,具有同样的生物学抗氧化功能及抗AS功效,而达到等效的抗AS效应,添加的茶粉影响了小鼠的体重增加;同时,与木瓜复合抗氧化物高剂量组添加有等量维生素C的单一纯维生素C添加组,并未显现出抗AS的生物学效应。因此,茶粉和维生素C的干预对比研究证明了重组木瓜抗氧化物在抗ApoE-/-小鼠动脉粥样硬化方面安全性更高、功效性更强。因此,本研究从木瓜抗氧化组分的重组技术着手,首先获得抗氧化物含量高、组分完整、稳定性强和生物利用率高的重组木瓜抗氧化物,然后添加入饲料中使动物以自然饮食方式进行补充,评价和探讨其对氧化损伤相关疾病——动脉粥样硬化的防治效应和机制,这不仅发挥了木瓜天然存在的抗氧化组分的复合抗氧化、抗炎症效应,从而具有抗AS生物学效应,还为后期推广到人群应用提供了实用的有效干预剂型。
Today it is widely believed that the development and progression of atherosclerosis is caused by a chronic inflammation in the vessel wall. Cells found in early atherosclerotic lesions are typically inflammatory cells (monocytes/ macrophages and T-lymphocytes). There is convincing clinical and experimental evidence that formation of reactive oxygen species (ROS) is augmented during this chronic inflammatory process due to an imbalance between synthesis of ROS and neutralizing antioxidative defense mechanisms. Biological molecules such as lipids, proteins and DNA may be reacted with ROS, which causes oxidative-stress induced damage. The evolving inflammatory reaction and ROS (any reactive organic or inorganic molecules with one or more unpairedelectrons) is instrumental in the initiation of atherosclerotic plaques and their destabilization Given that oxidative stress plays a pivotal role in the pathogenesis of cardiovascular diseases, it is not surprising that antioxidant therapies are one of the most effective and promising strategies against atherogenesis. Inhibition of ROS generation and function is a potential therapy to attenuate the extent of various cardiovascular diseases with nature antioidants ,such as bioflavonoids, which are polyphenolic compounds, are believed to be beneficial for the prevention and treatment of atherosclerosis and cardiovascular diseases because not only they possesses high antioxidant property and decreases the susceptibility of LDL to oxidation, but downregulate inflammatory factors (such as TNF-α) and attenuate accumulated macrophage-induced foam cells. There are abundant antioxidants, such as Vitamin C, polyphenolic compounds and superoxide dismutase in chaenomeles. With mice fed by extract of chaenomeles , Comparing to the model control group, the contents of MDA in serums and tissues of each dosages groups all decreased, and SOD activity in serums increased in irradiation peroxidation damage animal models. In vitro tissue factor inhibitory triterpene from the fruits of Chaenomeles sinensis was confirmed.
Consuming a diet rich in natural antioxidants or drug of synthesized antioxidants(probucol) have been associated with prevention from and/or treatment of atherosclerosis. Bioactive components of food with antioxidation, which are of special interest, include the Vitamins E and C, polyphenols, carotenoids and so on. Based on the‘oxidation theory’for atherosclerosis, dietary antioxidants have attracted considerable attention as preventive and therapeutic agents. There is adequate evidence from observational, in vitro, ex vivo, controlled intervention and animal model studies that consumption of certain foods results to a reduction in oxidative stress and myocardial infarction biomarkers. The effects of dietary antioxidants assessed in vitro, ex vivo and in vivo was shown below: (1) Increase in plasma oxidative resistance;(2) Decrease in plasma MDA levels;(3) Increase in LDL oxidative resistance;(4) Decrease in foam cell formation and extend of the atherosclerotic lesion; (5) Upregulation of GSH-Px, SOD, catalase gene expression; (6) Decrease in macrophage accumulation and severity of the lesion in the aorta;(7) Suppression of TNF-αmRNA expression. The antioxidant properties of plant components, such as carotenoids, vitamin C, flavonoid, are individually examined by epidemiological studies or supplementation trials. However, the results are not as clear-cut as those obtained for fruit and vegetables and are often disappointing. Since there is an agreement that not one antioxidant alone can lead to health benefits but the combination, as found for example in fruits and vegetables, is the active principle, transferred the main antioxidative composition from the fruit of Chaenomeles speciosa into maltodextrin (Chaenomeles speciosa powder, CSP) by process with a combination of enzyme-aided extraction and fluid bed spray-drying techniques, to preserve their antioxidant activity. Subsequently, we determined the contents of antioxidants and antioxidant capacity, and further to evaluated the potential anti-atherosclerotic effects of CSP in apolipoprotein E deficient (ApoE-/-) mice. Method:
Chaenomeles (Cultivated in Qijiang county of Chonqing, China) that have been frozen, pulped with distilled water, cured with enzymes (the pectinase and cellulase), spray-dried with maltodextrin and reground. To assess the effects of enzymolysis on the juice yield of the extraction, the conditions for enzymolysis, including concentrations of pectinase and cellulase, temperature and time, were investigated through an orthogonal design of L9(34) on juice yield. The aqueous extract by optimal enzymatic technique was processed to CSP by techniques of inclusion and delayed release powder granulated. Subsequently, the contents of antioxidants (VitaminC, total phenols, tannin) and antioxidant capacity(TAC, SOD and FRAP ) were determined. Polyphenol compunds were analyzed by TLC for the qualitation of the monomers and tannin. Microplate quantification of DPPH?was used to estimate the change of antioxidant system in chaenomeles and CSP by process with comparison of kinetic curve.
Apoe-/- mice of age 6 wk and weight 20±3 g were kindky provided by Pr. Ling at Sun Yat-Sen University. All animal procedures were followed in accordance with the approved protocol for use of experimental animals set by the standing committee on animal care at The Third Military Medical University. Mice were averagely divided into five groups and then fed with 5g CSP/100g high-fat diet (low dosage, 5%CSP group) ,with 10g CSP/100g high-fat diet (high dosage, 10%CSP group) , with 3 g green tea powder high-fat diet (Tea group), with 0.02% Vitamin C high-fat diet (VC group)for 16 wk and fed high-fat diet without CSP were used as control . All mice were then killed by decapitation under anesthesia. The blood samples were harvested for serum lipid analysis and antioxidant capacity determanation, and the descending aorta, aortic sinus and aortic arch were collected for quantification analysis of lipidoses, atherosclerotic plaque area and intima-media thichness by staining with oil red and hematoxilin-eosin, respectively. The expression of TNF-α, CD68 and the amount of macrophagocyte were determined by immunohistochemistry. The histopathological studies of blood vessel endothelium were carried by electron microscope scanning.
Results
Enzymolysis technique was optimized as 2.0 ml/100g of pectinase and 2.0 ml/100g of cellulase in water bath at 40℃for 60 min. The technique of Fluid Bed Granulation is that: Temperature control in batch at 45℃, frequency of aerator at 4~5kHz, peristaltic pump at 3~5Hz, the frequency of buffeting at 35~40 times/min There were 2.05±0.09mg Vitamin C, 22.70±1.57mg total phenols and 9.64±0.65 mg tannin in 1g CSP. There are rutin, catechin, quercetin, chlorogenic acid and procyanidine. The ferric reducing antioxidant power of CSP was (172.9±6.7)μmol Fe2+/g and the scavenging activity on DPPH?(TAC) and O2 ? (SOD) were (945±20)μg DPPH?/g and (700±21) U, respectively. there was no much change of antioxidant compound and property by process with confirmation of MQ method. The associated binding constants of enzymatic extract withγ-cyclodextrin.
Comparing with control group, A dose-dependent inhibitory effect of CSP on reduction of serum LDL cholesterol and TC levels (P<0.05). There was a reduction of TG found in10%CSP and tea groups. Comparing with control group Tea, 5% and 10% CSP dietary supplement significantly increased glutathione peroxidase activity and total anti-oxidative capacity, and subsequently achieved a reduction of lipidoses on descending aorta by 29.2%, 40.8% and 67.3%; reduction of IMT by 35.1%, 30.1% and 48.2%; reduction of relative atherosclerotic plaque area of aortic sinus by 17.4%, 23.9% and 38.1% in ApoE-/- mice. Concerning about inflammatory factor, tea and CSP significantly downregulated the expression TNF-αand CD68, especially a dose-dependent effect of CSP. There were also amounts of macrophagocyte decreased significantly by tea and CSP supplement(P<0.05).
Conclusion:
We successfully transferred antioxidants in Chaenomeles to the powder composed of cyclodextrin with good shelf life, achieving abundant Vitamin C and polyphenols, which achieved scavenging superoxide radical and DPPH?. CSP significantly increase antioxidant status of plasma, which would enhance LDL oxidative resistance; The decrease of serum TG, TC, LDL would reduce lipidoses and subsequently attenuate foam cell formation and extend of the atherosclerotic lesion; The downregulation of expression of TNF-αand CD68 would reflect lower level of inflammation, which decrease in macrophage accumulation and severity of the lesion in the plague. In summary, as reported that increased levels of LDL together with decreased antioxidant capacity of blood may accelerate the formation of atherosclerosis by increasing LDL-oxidation and ox-LDL is atherogenic, the anti-atherosclerotic of the powder processed by Chaenomeles Speciosa was maybe due to the enhanced antioxidative status in circulation, anti-inflammation in the aorta endothelium and the decreased serum cholesterol levels, especially LDL.
目前对于通过抗氧化效应防治AS的研究主要关注的抗氧化物类别是维生素类、植物化合物及人工合成的抗氧化剂,多见于维生素E、维生素C、植物黄酮、原花青素和普罗布考等。将其中具有阳性结果的研究所涉及到的防治AS机制进行总结,有如下几个方面:(1)提高了血清抗氧化还原能力(T-AOC);(2)降低了血清MDA的水平;(3)抑制了LDL的氧化;(4)降低了泡沫样细胞的形成;(5)上调了GSH-Px、SOD、过氧化氢酶的基因表达;(6)下调了巨噬细胞的聚集程度;(7)抑制了TNF-α基因的表达。这些抗氧化剂最终表现出来的抗AS组织病理学变化是中内膜厚度(IMT)最大值减小和斑块尺寸缩小。但不少的单一抗氧化剂干预AS实验并没有达到预期的结果,有越来越多的研究者认为抗氧化物之间的协同效应发挥非常重要,而以植物粗提物或某一大类复合抗氧化组分来研究抗AS作用所获得的实验结果越来越振奋人心。
因此,本研究选用含丰富抗氧化组分的木瓜(Chaenomeles speciosa)为原料,采用生物酶解、环糊精包合和喷雾制粒技术,将存在于植物体组织中的抗氧化物质抽提出来,对其进行包合修饰,重新将其固化在环糊精给药载体上,将这种重新组合(下简称:重组)的木瓜复合抗氧化物添加入高脂饲料中,喂饲ApoE基因缺陷(ApoE-/-)小鼠,对木瓜抗氧化体系防治AS的生物学效应及机制进行研究和探讨。
方法:
采用纤维素-果胶酶处理木瓜,通过正交实验设计获取最优酶解工艺;对提取的木瓜抗氧化复合物中的多酚类物质进行TLC薄层色谱鉴定,采用λ-环糊精包合技术对酶解提取的木瓜复合抗氧化物进行包合修饰;采用流化床喷雾制粒技术制备由β-环糊精为主要载体的重组木瓜复合抗氧化物颗粒(CSP);比较木瓜果实及CSP中维生素C含量、总酚量、单宁量以及FRAP、SOD的活性;采用改良的DPPH微孔板定量法(MQ)评测加工重组前后总抗氧化能力(TAC)的变化,并结合动力学曲线分析木瓜抗氧化体系重组前后其抗氧化特征变化。选用ApoE-/-雄性2月龄小鼠40只,随机分为5组,分别为空白对照组(Control)、CSP低剂量组(5%CSP)、CSP高剂量组(10%CSP)、超微茶粉组(Tea)、维生素C组(VC),在SPF级动物房喂养16周,眼眶取血测定血脂(TC、TG、LDL-C、HDL-C)、血清总抗氧化能力(T-AOC)和血清谷胱甘肽过氧化物酶(GSH-Px);免疫组化测定斑块TNF-α表达水平,免疫荧光检测CD68表达水平和巨噬细胞计数;HE染色分析主动脉窦斑块面积率及主动脉弓中内膜厚度(IMT);油红O染色降主动脉分析脂质沉积血管内膜状况,结合电镜扫描阐述动脉血管内壁微观变化。
主要结果和结论如下:
1.酶解处理木瓜果实体显著提高了木瓜出汁率,其最优酶解工艺为果胶酶添加量2%,纤维素酶添加量2%,温度40℃,时间60分钟。酶解技术的运用使木瓜主要抗氧化物——维生素C、总酚、单宁和黄酮提取总量分别提高了35.1%、43.8%、45.3%和46.7%。通过TLC色谱鉴定,木瓜提取物中含有儿茶素、表儿茶素和槲皮素、芦丁、绿原酸和原花青素。酶解使木瓜组织细胞壁崩解,这有利于完整地将木瓜原有的抗氧化组分浸提出来、重组到环糊精给药载体上。
2.γ-环糊精通过单一物质包合反应验证了其对木瓜含有的维生素C(呋喃烯醇式内酯结构)、原花青素(单宁类多聚体结构)、绿原酸(缩酚酸结构)和芦丁(类黄酮单元结构)具有包合作用。多酚类物质的包合常数在2.8×103～4.2×104之间,其Ka随多酚物质分子量增加而增大;γ-环糊精对维生素C的包合常数也达到了5.7×103。木瓜酶解提取复合物与γ-环糊精反应达到平衡时的包合常数为1.9×103,表明包合反应充分。包合修饰的实现有助于后期重组固化制粒的加工稳定性、耐储藏性及生物利用率的提高。
3.采用顶喷流化床喷雾制粒工艺,流化床体中心温度45℃、风机频率4~5kHz、进料泵3~5Hz、抖料器频率35~40次/min;获得的重组木瓜复合抗氧化物(CSP)每克含有维生素C、总酚、单宁含量分别为2.05±0.09mg、22.70±1.57mg和9.64±0.65mg,比重组前的木瓜抗氧化体系单位质量含有的维生素C、总酚和单宁量提高了219.8%、344.2%、348.4%;每克CSP的SOD活性、FRAP和TAC分别为700±21U、172.9±6.7μmol Fe2+、945±20μg DPPH?,比重组前的木瓜抗氧化体系单位质量的SOD活性、FRAP和TAC提高了236.5%、213.8%、300.4%。重组的木瓜抗氧化体系不仅提高了抗氧化物的含量,还比木瓜果实中抗氧化体系在储藏过程中更稳定,更易于在后期动物干预过程中保持应有的抗氧化活性。
4.改良创新了DPPH?微孔板测定技术(MQ)测定植物总抗氧化能力(TAC),不仅具有批处理量大、节约试剂和减少系统误差的优点,还能同时完成对抗氧化组分物质清除自由基反应动力学特征的测定。通过MQ评测木瓜抗氧化体系重组加工前后TAC及动力学曲线特征,推断重组后的木瓜抗氧化体系没有改变原有的抗氧化特性。
5.木瓜复合抗氧化物(CSP)在抗ApoE-/-小鼠动脉粥样硬化的生物学效应方面,其组织病理学变化主要表现为:CSP显著减少了降主动脉管壁内表面的脂质沉积发生,5%和10%的CSP干预后ApoE-/-小鼠降主动脉管壁内表面的脂质沉积面积率(OSL)分别为0.22和0.12;对比Control组,5%、10%的CSP添加分别使ApoE-/-小鼠降主动脉管壁内表面脂质沉积减少了40.8%和67.3%。CSP显著抑制了主动脉弓中内膜增厚的病变,低剂量和高剂量CSP干预后的ApoE-/-小鼠的主动脉弓中内膜厚度均值(IMT)分别为128.9和95.5;对比Control组,5%、10%的CSP添加分别减少了ApoE-/-小鼠主动脉弓中内膜30.1%和48.2%的增厚量。CSP显著减小了主动脉窦斑块的尺寸,5%和10%的CSP干预后ApoE-/-小鼠的主动脉窦斑块面积率分别为0.339和0.276;对比Control组,添加5%的CSP使ApoE-/-小鼠的主动脉窦斑块缩小了23.1%,添加10%的CSP使ApoE-/-小鼠的主动脉窦斑块缩小了38.1%。实验表明重组的木瓜抗氧化物从AS的发生阶段——“脂质沉积”到AS的发展阶段——“斑块形成”,都表现出了抗AS的生物学效应。
6.木瓜复合抗氧化物(CSP)抗AS的相关机制实验研究结果及结论:
1)木瓜复合抗氧化物显著降低了ApoE-/-小鼠的TC、TG、LDL-C水平。对比Control组血脂指标水平,添加5%的CSP分别使ApoE-/-小鼠血清TC和LDL-C降低了14%和21.2%;添加10%的CSP分别使ApoE-/-小鼠血清TC和LDL-C降低了17.3%和46.6%,而且还观察到了TG有36.6%的下降。实验结果充分表明重组的木瓜复合抗氧化物具有降低诱发AS相关血脂指标水平的作用,特别是降低主要风险因素——LDL-C的血清浓度。
2)木瓜复合抗氧化物显著提高了血清抗氧化的能力。对比Control组的血清抗氧化指标,添加5%的CSP分别使ApoE-/-小鼠血清GSH-PX和T-AOC提高了30%和53.3%;添加10%的CSP分别使ApoE-/-小鼠血清GSH-PX和T-AOC提高了47.1%和126.7%。实验结果表明,重组的复合木瓜抗氧化物不仅在体外FRAP、SOD和TAC抗氧化指标上表现出很强的抗氧化特性,还在体内发挥出了提高血清抗氧化能力的生物学抗氧化效应,这对AS发生发展的关键风险因子——ox-LDL的生成有潜在地抑制作用。
3)木瓜复合抗氧化物显著下调了ApoE-/-小鼠主动脉窦斑块中TNF-α的表达。以Control组主动脉斑块中TNF-α评分结果为基准,添加5%、10%的CSP使ApoE-/-小鼠主动脉窦TNF-α分值分别降低了19%和67%。实验结果表明重组的木瓜复合抗氧化物具有抗炎生物学效应,这对由炎性因子介导的AS发生发展效应有潜在的抑制作用。
4)木瓜复合抗氧化物显著下调了ApoE-/-小鼠主动脉窦部CD68的表达水平和减少了巨噬细胞数。以Control组免疫荧光结果为基准,添加5%、10%的CSP分别使ApoE-/-小鼠主动脉窦部巨噬细胞标记物CD68表达水平下调了25.1%和38.3%;添加5%、10%的CSP分别使ApoE-/-小鼠主动脉窦部巨噬细胞数减少了42.8%和50.3%。实验结果表明,重组的木瓜复合抗氧化物对巨噬细胞介导的AS促发效应有潜在的抑制作用。
5)通过电镜扫描观察,ApoE-/-小鼠实验模型有典型的AS发生发展表征,如脂质沉积引发了血管壁表面微观结构的变化,表现为内膜纹理走向紊乱、表面有“虫蚀”样、间隙增大,血管壁向管腔隆起增厚和血小板凝集变形沉积在管壁上。这些都与本实验探究的血脂异常、炎性因子水平、巨噬细胞数和机体抗氧化状态有密切联系和相关性。
7.重组的木瓜复合抗氧化物比发挥相似抗AS效应的茶粉添加来说,对ApoE-/-小鼠生长影响更小,干预过程更安全;重组的木瓜复合抗氧化颗粒比单一维生素C添加对ApoE-/-小鼠的AS防治更有效。
综上所述,本研究通过酶解提取、包合修饰和喷雾制粒技术将木瓜中的抗氧化组分成功地、较完整地重组到了环糊精给药载体上,制备成了具有良好储藏特性的木瓜复合抗氧化颗粒,其含有丰富的维生素C、多酚类物质,具有清除超氧阴离子、脂质自由基和铁离子还原能力;通过ApoE-/-小鼠动脉粥样硬化干预实验模型证实了重组的木瓜复合抗氧化物具有防治动脉粥样硬化的功效,主要体现为减少降主动脉内壁脂质沉积、抑制主动脉弓中内膜厚度的增加、抑制主动脉窦斑块的增长。其可能的机制是木瓜抗氧化物通过降低血清TG、TC和LDL-C的浓度水平、提高机体抗氧化能力(血清GSH-PX和T-AOC的增加)从而抑制LDL的氧化程度、降低炎性因子的表达及炎性细胞的数量发挥抗动脉粥样硬化的生物学效应。实验还通过对比研究添加超微绿茶粉(多酚类抗氧化物为主的抗氧化剂)和维生素C对ApoE-/-小鼠动脉粥样硬化的防治功效,证实了含有总酚量相近的超微绿茶粉在很多生物学效应上与添加木瓜复合抗氧化物的低剂量组一致,从一个侧面证实了木瓜复合抗氧化物中的多酚类物质与目前研究公认的防心血管疾病茶多酚类物质,具有同样的生物学抗氧化功能及抗AS功效,而达到等效的抗AS效应,添加的茶粉影响了小鼠的体重增加;同时,与木瓜复合抗氧化物高剂量组添加有等量维生素C的单一纯维生素C添加组,并未显现出抗AS的生物学效应。因此,茶粉和维生素C的干预对比研究证明了重组木瓜抗氧化物在抗ApoE-/-小鼠动脉粥样硬化方面安全性更高、功效性更强。因此,本研究从木瓜抗氧化组分的重组技术着手,首先获得抗氧化物含量高、组分完整、稳定性强和生物利用率高的重组木瓜抗氧化物,然后添加入饲料中使动物以自然饮食方式进行补充,评价和探讨其对氧化损伤相关疾病——动脉粥样硬化的防治效应和机制,这不仅发挥了木瓜天然存在的抗氧化组分的复合抗氧化、抗炎症效应,从而具有抗AS生物学效应,还为后期推广到人群应用提供了实用的有效干预剂型。
Today it is widely believed that the development and progression of atherosclerosis is caused by a chronic inflammation in the vessel wall. Cells found in early atherosclerotic lesions are typically inflammatory cells (monocytes/ macrophages and T-lymphocytes). There is convincing clinical and experimental evidence that formation of reactive oxygen species (ROS) is augmented during this chronic inflammatory process due to an imbalance between synthesis of ROS and neutralizing antioxidative defense mechanisms. Biological molecules such as lipids, proteins and DNA may be reacted with ROS, which causes oxidative-stress induced damage. The evolving inflammatory reaction and ROS (any reactive organic or inorganic molecules with one or more unpairedelectrons) is instrumental in the initiation of atherosclerotic plaques and their destabilization Given that oxidative stress plays a pivotal role in the pathogenesis of cardiovascular diseases, it is not surprising that antioxidant therapies are one of the most effective and promising strategies against atherogenesis. Inhibition of ROS generation and function is a potential therapy to attenuate the extent of various cardiovascular diseases with nature antioidants ,such as bioflavonoids, which are polyphenolic compounds, are believed to be beneficial for the prevention and treatment of atherosclerosis and cardiovascular diseases because not only they possesses high antioxidant property and decreases the susceptibility of LDL to oxidation, but downregulate inflammatory factors (such as TNF-α) and attenuate accumulated macrophage-induced foam cells. There are abundant antioxidants, such as Vitamin C, polyphenolic compounds and superoxide dismutase in chaenomeles. With mice fed by extract of chaenomeles , Comparing to the model control group, the contents of MDA in serums and tissues of each dosages groups all decreased, and SOD activity in serums increased in irradiation peroxidation damage animal models. In vitro tissue factor inhibitory triterpene from the fruits of Chaenomeles sinensis was confirmed.
Consuming a diet rich in natural antioxidants or drug of synthesized antioxidants(probucol) have been associated with prevention from and/or treatment of atherosclerosis. Bioactive components of food with antioxidation, which are of special interest, include the Vitamins E and C, polyphenols, carotenoids and so on. Based on the‘oxidation theory’for atherosclerosis, dietary antioxidants have attracted considerable attention as preventive and therapeutic agents. There is adequate evidence from observational, in vitro, ex vivo, controlled intervention and animal model studies that consumption of certain foods results to a reduction in oxidative stress and myocardial infarction biomarkers. The effects of dietary antioxidants assessed in vitro, ex vivo and in vivo was shown below: (1) Increase in plasma oxidative resistance;(2) Decrease in plasma MDA levels;(3) Increase in LDL oxidative resistance;(4) Decrease in foam cell formation and extend of the atherosclerotic lesion; (5) Upregulation of GSH-Px, SOD, catalase gene expression; (6) Decrease in macrophage accumulation and severity of the lesion in the aorta;(7) Suppression of TNF-αmRNA expression. The antioxidant properties of plant components, such as carotenoids, vitamin C, flavonoid, are individually examined by epidemiological studies or supplementation trials. However, the results are not as clear-cut as those obtained for fruit and vegetables and are often disappointing. Since there is an agreement that not one antioxidant alone can lead to health benefits but the combination, as found for example in fruits and vegetables, is the active principle, transferred the main antioxidative composition from the fruit of Chaenomeles speciosa into maltodextrin (Chaenomeles speciosa powder, CSP) by process with a combination of enzyme-aided extraction and fluid bed spray-drying techniques, to preserve their antioxidant activity. Subsequently, we determined the contents of antioxidants and antioxidant capacity, and further to evaluated the potential anti-atherosclerotic effects of CSP in apolipoprotein E deficient (ApoE-/-) mice. Method:
Chaenomeles (Cultivated in Qijiang county of Chonqing, China) that have been frozen, pulped with distilled water, cured with enzymes (the pectinase and cellulase), spray-dried with maltodextrin and reground. To assess the effects of enzymolysis on the juice yield of the extraction, the conditions for enzymolysis, including concentrations of pectinase and cellulase, temperature and time, were investigated through an orthogonal design of L9(34) on juice yield. The aqueous extract by optimal enzymatic technique was processed to CSP by techniques of inclusion and delayed release powder granulated. Subsequently, the contents of antioxidants (VitaminC, total phenols, tannin) and antioxidant capacity(TAC, SOD and FRAP ) were determined. Polyphenol compunds were analyzed by TLC for the qualitation of the monomers and tannin. Microplate quantification of DPPH?was used to estimate the change of antioxidant system in chaenomeles and CSP by process with comparison of kinetic curve.
Apoe-/- mice of age 6 wk and weight 20±3 g were kindky provided by Pr. Ling at Sun Yat-Sen University. All animal procedures were followed in accordance with the approved protocol for use of experimental animals set by the standing committee on animal care at The Third Military Medical University. Mice were averagely divided into five groups and then fed with 5g CSP/100g high-fat diet (low dosage, 5%CSP group) ,with 10g CSP/100g high-fat diet (high dosage, 10%CSP group) , with 3 g green tea powder high-fat diet (Tea group), with 0.02% Vitamin C high-fat diet (VC group)for 16 wk and fed high-fat diet without CSP were used as control . All mice were then killed by decapitation under anesthesia. The blood samples were harvested for serum lipid analysis and antioxidant capacity determanation, and the descending aorta, aortic sinus and aortic arch were collected for quantification analysis of lipidoses, atherosclerotic plaque area and intima-media thichness by staining with oil red and hematoxilin-eosin, respectively. The expression of TNF-α, CD68 and the amount of macrophagocyte were determined by immunohistochemistry. The histopathological studies of blood vessel endothelium were carried by electron microscope scanning.
Results
Enzymolysis technique was optimized as 2.0 ml/100g of pectinase and 2.0 ml/100g of cellulase in water bath at 40℃for 60 min. The technique of Fluid Bed Granulation is that: Temperature control in batch at 45℃, frequency of aerator at 4~5kHz, peristaltic pump at 3~5Hz, the frequency of buffeting at 35~40 times/min There were 2.05±0.09mg Vitamin C, 22.70±1.57mg total phenols and 9.64±0.65 mg tannin in 1g CSP. There are rutin, catechin, quercetin, chlorogenic acid and procyanidine. The ferric reducing antioxidant power of CSP was (172.9±6.7)μmol Fe2+/g and the scavenging activity on DPPH?(TAC) and O2 ? (SOD) were (945±20)μg DPPH?/g and (700±21) U, respectively. there was no much change of antioxidant compound and property by process with confirmation of MQ method. The associated binding constants of enzymatic extract withγ-cyclodextrin.
Comparing with control group, A dose-dependent inhibitory effect of CSP on reduction of serum LDL cholesterol and TC levels (P<0.05). There was a reduction of TG found in10%CSP and tea groups. Comparing with control group Tea, 5% and 10% CSP dietary supplement significantly increased glutathione peroxidase activity and total anti-oxidative capacity, and subsequently achieved a reduction of lipidoses on descending aorta by 29.2%, 40.8% and 67.3%; reduction of IMT by 35.1%, 30.1% and 48.2%; reduction of relative atherosclerotic plaque area of aortic sinus by 17.4%, 23.9% and 38.1% in ApoE-/- mice. Concerning about inflammatory factor, tea and CSP significantly downregulated the expression TNF-αand CD68, especially a dose-dependent effect of CSP. There were also amounts of macrophagocyte decreased significantly by tea and CSP supplement(P<0.05).
Conclusion:
We successfully transferred antioxidants in Chaenomeles to the powder composed of cyclodextrin with good shelf life, achieving abundant Vitamin C and polyphenols, which achieved scavenging superoxide radical and DPPH?. CSP significantly increase antioxidant status of plasma, which would enhance LDL oxidative resistance; The decrease of serum TG, TC, LDL would reduce lipidoses and subsequently attenuate foam cell formation and extend of the atherosclerotic lesion; The downregulation of expression of TNF-αand CD68 would reflect lower level of inflammation, which decrease in macrophage accumulation and severity of the lesion in the plague. In summary, as reported that increased levels of LDL together with decreased antioxidant capacity of blood may accelerate the formation of atherosclerosis by increasing LDL-oxidation and ox-LDL is atherogenic, the anti-atherosclerotic of the powder processed by Chaenomeles Speciosa was maybe due to the enhanced antioxidative status in circulation, anti-inflammation in the aorta endothelium and the decreased serum cholesterol levels, especially LDL.
引文
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1. Peter Libby and Paul M. Ridker, Inflammation and Atherothrombosis: From Population Biology and Bench Research to Clinical Practice. Journal of the American College of Cardiology, 2006. 48(9, Supplement 1): p. A33-A46.
2. Russell Ross, Atherosclerosis -- An Inflammatory Disease. N Engl J Med, 1999. 340(2): p. 115-126.
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5. Jens Milde., Erich F. Elstner., and Johanna Grassmann., Synergistic effects of phenolics and carotenoids on human low-density lipoprotein oxidation. Molecular Nutrition & Food Research, 2007. 51(8): p. 956-961.
6. B. Fuhrman., et al., Lycopene synergistically inhibits LDL oxidation in combination with vitamin E, glabridin, rosmarinic acid, carnosic acid, or garlic. Antioxidants & Redox Signaling 2000. 2(3 ): p. 491-506.
7. Yamakoshi J, Kataoka S, and Koga T, Proanthocyanidinsrich extract from grape seeds attenuates the development of aortic atheroselerosis in cholesterol-fedrabbits. Atherosclerosis, 1999. 142: p. 139-149.
8. Naghma Khan and Hasan Mukhtar, Tea polyphenols for health promotion. Life Sciences, 2007. 81(7): p. 519-533.
9. Sung I. Koo and Sang K. Noh, Green tea as inhibitor of the intestinal absorption of lipids: potential mechanism for its lipid-lowering effect. The Journal of Nutritional Biochemistry, 2007. 18(3): p. 179-183.
10. Sano M, Effect of tea on lipid peroxidation in rat liver and kidney :a comparison of green and black tea feeding. Biolpharm Bull, 1995. 18(7): p. 1006～1008.
11. Nurulain T. Zaveri, Green tea and its polyphenolic catechins: Medicinal uses in cancer and noncancer applications, in Life Sciences;NATURECEUTICALS (NATURAL PRODUCTS), NUTRACEUTICALS, HERBAL BOTANICALS, AND PSYCHOACTIVES: DRUG DISCOVERY AND DRUG-DRUG INTERACTIONS - Volume II: Nutraceuticals, Herbals and Related Products. 2006. p. 2073-2080.
2. Katia Tebib, et al., Polymeric grape seed tannins prevent plasma cholesterol changes in high-cholesterol-fed rats. Food Chemistry, 1994. 49(4): p. 403-406.
3. Anis Arnous, Dimitris P. Makris, and Panagiotis Kefalas., Effect of Principal Polyphenolic Components in Relation to Antioxidant Characteristics of Aged Red Wines. Agric. Food Chem, 2001. 49(12): p. 5736 -5742.
4. Keyvan Dastmalchi, et al., Chemical composition and in vitro antioxidant evaluation of a water-soluble Moldavian balm (Dracocephalum moldavica L.) extract. LWT - Food Science and Technology, 2007. 40(2): p. 239-248.
5. Wieland Peschel, et al., An industrial approach in the search of natural antioxidants from vegetable and fruit wastes, Food Chemistry;. 2006. p. 137-150.
6. John D. Potter, Cancer prevention: epidemiology and experiment. Cancer Letters, 1997. 114(1-2): p. 7-9.
7. Yanping Zou, Yanhua Lu, and Dongzhi Wei, Hypocholesterolemic effects of a flavonoid-rich extract of Hypericum perforatum L. in rats fed a cholesterol-rich diet. J. Agric. Food Chem, 2005. 53(7): p. 2462 -2466.
8. A. C. Kaliora, G. V. Z. Dedoussis, and H. Schmidt, Dietary antioxidants in preventing atherogenesis. Atherosclerosis, 2006. 187(1): p. 1-17.
9. L. P. Leong and G. Shui, An investigation of antioxidant capacity of fruits in Singapore markets, in Food Chemistry. 2002. p. 69-75.
10. I. Krinsky Norman, Carotenoids as antioxidants, in Nutrition. 2001. p. 815-17.
11. Ntei Abudu, et al., Vitamins in human arteriosclerosis with emphasis on vitamin C and vitamin E, in Clinica Chimica Acta;. 2004. p. 11-25.
12. Yusuf Ozkan, et al., Effects of triple antioxidant combination (vitamin E, vitamin C and [alpha]-lipoic acid) with insulin on lipid and cholesterol levels and fatty acid composition of brain tissue in experimental diabetic and non-diabetic rats, Cell Biology International;. 2005. p. 754-760.
13. K. Imao, et al., Free radical scavenging activity of fermented papaya preparation andits effect on lipid peroxide level and superoxide dismutase activity in iron-induced epileptic foci of rats, Biochem Mol Biol Int. 1998: AUSTRALIA. p. 11-23.
14. Josefa M. Navarro, et al., Changes in the contents of antioxidant compounds in pepper fruits at different ripening stages, as affected by salinity, Food Chemistry;. 2006. p. 66-73.
15.房喻,代郡和吕卫明,酶水解法提取分离黄芩素的研究,中国中药杂志. 1991. p. 742.
16.汤海鸥,黑曲霉酶解提取葛根黄酮的研究,饲料工业. 2006. p. 14-18.
17. Ourdia Bouzid, et al., Fungal enzymes as a powerful tool to release simple phenolic compounds from olive oil by-product, Process Biochemistry;. 2005. p. 1855-1862.
18. Chang-Bum Ahn, et al., Free radical scavenging activity of enzymatic extracts from a brown seaweed Scytosiphon lomentaria by electron spin resonance spectrometry, Food Research International;. 2004. p. 253-258.
19. I. Orienti, et al., Complexation of ursodeoxycholic acid with [beta]-cyclodextrin-choline dichloride coprecipitate, in International Journal of Pharmaceutics;. 1999. p. 139-153.
20. M. L. Calabro, et al., Effects of [alpha]- and [beta]-cyclodextrin complexation on the physico-chemical properties and antioxidant activity of some 3-hydroxyflavones, Journal of Pharmaceutical and Biomedical Analysis;10th Internat. 2004. p. 365-377.
21. Pung Sok Lee, et al., Physicochemical characteristics and bioavailability of a novel intestinal metabolite of ginseng saponin (IH901) complexed with [beta]-cyclodextrin, International Journal of Pharmaceutics;. 2006. p. 29-36.
22. Francesco Cipollone, Maria Luigia Fazia, and Andrea Mezzetti, Oxidative stress, inflammation and atherosclerotic plaque development. International Congress Series, 2007. 1303: p. 35-40.
23. Russell Ross, Atherosclerosis -- An Inflammatory Disease. N Engl J Med, 1999. 340(2): p. 115-126.
24. Luigi Giusto Spagnoli, et al., Role of Inflammation in Atherosclerosis. J Nucl Med, 2007. 48(11): p. 1800-1815.
25.祝杰和凌文华,氧化、抗氧化与动脉粥样硬化,疾病控制杂志. 2001. p. 338-340.
26. Michael Aviram and Bianca Fuhrman, Polyphenolic flavonoids inhibit macrophage-mediated oxidation of LDL and attenuate atherogenesis. Atherosclerosis, 1998. 137(Supplement 1): p. S45-S50.
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6. B. Fuhrman., et al., Lycopene synergistically inhibits LDL oxidation in combination with vitamin E, glabridin, rosmarinic acid, carnosic acid, or garlic. Antioxidants & Redox Signaling 2000. 2(3 ): p. 491-506.
7. Yamakoshi J, Kataoka S, and Koga T, Proanthocyanidinsrich extract from grape seeds attenuates the development of aortic atheroselerosis in cholesterol-fedrabbits. Atherosclerosis, 1999. 142: p. 139-149.
8. Naghma Khan and Hasan Mukhtar, Tea polyphenols for health promotion. Life Sciences, 2007. 81(7): p. 519-533.
9. Sung I. Koo and Sang K. Noh, Green tea as inhibitor of the intestinal absorption of lipids: potential mechanism for its lipid-lowering effect. The Journal of Nutritional Biochemistry, 2007. 18(3): p. 179-183.
10. Sano M, Effect of tea on lipid peroxidation in rat liver and kidney :a comparison of green and black tea feeding. Biolpharm Bull, 1995. 18(7): p. 1006～1008.
11. Nurulain T. Zaveri, Green tea and its polyphenolic catechins: Medicinal uses in cancer and noncancer applications, in Life Sciences;NATURECEUTICALS (NATURAL PRODUCTS), NUTRACEUTICALS, HERBAL BOTANICALS, AND PSYCHOACTIVES: DRUG DISCOVERY AND DRUG-DRUG INTERACTIONS - Volume II: Nutraceuticals, Herbals and Related Products. 2006. p. 2073-2080.
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