【作者】 韩江南；

【导师】 马海乐； 樊金铃；

【作者基本信息】 河南科技大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 牡丹为我国著名花卉,被称为“万花一品”、“冠绝群芳”的“花王”。花色是牡丹重要的观赏性状,花色素则是牡丹花色形成的物质基础,又因多种生理活性而具有潜在的应用价值。本文主要研究了中原牡丹品种群的花色、花色苷及二者的相关性,确定了大孔吸附树脂法纯化牡丹花色苷的工艺条件,测定了牡丹花提取液与花色苷的抗氧化活性。具体研究内容及结果如下:1 .利用色差仪按国际照明委员会( International Commission on Illumination,CIE)表色系统对94个中原牡丹品种的花色进行测定,并在此基础上,通过CLUSTER过程将花色三刺激值(明度L*、色相a*和b*)采用Ward离差平方和法进行了聚类分析。结果表明:94个中原牡丹品种花色在CIE表色系统坐标系上的分布广泛,聚类分析将其分为9个色系:白(20种)、绿(1种)、浅粉(8种)、粉红(13种)、粉蓝(12种)、红(14种)、紫(11种)、红紫(7种)、红黑(8种),不同色系牡丹的L*、a*和b*值特征明显。白、浅粉、粉红和红色系以及粉蓝、紫和红紫色系的L*与a*、L*与彩度(C*)均表现出显著的负相关性(L*与a*的相关系数R2分别为0.9703和0.6067,L*与C*的相关系数R2分别为0.8519和0.714);多数色系牡丹(除红和红黑外)的L*与b*呈显著的正相关关系(R2为0.8653)。2.采用高效液相色谱与二极管陈列检测器和电喷雾电离质谱联用技术(HPLC-DAD-ESI-MS)分析了48个品种的中原牡丹花瓣中的花色苷总含量、种类以及各花色苷单体的相对丰度。结果表明:中原牡丹花中共含有基于天竺葵色素、芍药色素和矢车菊色素的五种花色苷,分别为矢车菊色素-3, 5-O-二葡萄糖苷(Cy3G5G),矢车菊色素-3-O-葡萄糖苷(Cy3G)、芍药色素-3, 5-O-二葡萄糖苷(Pn3G5G)、芍药色素-3-O-葡萄糖苷(Pn3G)和天竺葵色素-3, 5-O-二葡萄糖苷(Pg3G5G),不含天竺葵色素-3-O-葡萄糖苷(Pg3G)。48个品种中原牡丹花色苷总含量变化范围为0~289.5mg/100g;不同色系牡丹花色苷单体的相对丰度差异显著,粉红和红色系品种以天竺葵色素-3, 5- O-二葡萄糖苷为主(相对丰度分别为88.09%和79.06%),不含或含有近少量Pn类色素,属于PgPn类;粉蓝、紫和红黑、红紫色系以芍药色素-3, 5- O-二葡萄糖苷为主(相对丰度分别为88.35%、82.19%和75.65%、72.74%),矢车菊类色素在红紫和红黑色系牡丹中的相对丰度较大,属于PnCy类。通过分析牡丹花色与花色苷关系可知,亮度值L*与花色苷总量(TA)有显著负相关性,在PnCy类中a*和b*都与TA没有相关性,而PnPg类a*与TA正相关,b*与TA没有相关性。3.研究比较了多种大孔吸附树脂的静态吸附特性,筛选出了适宜牡丹花色苷纯化的树脂,并确定了固定床吸附和解吸的工艺参数。结果表明:南开化工D-101、天津大钧D-401和安徽三星D-1300三种树脂的吸附率和解析率较高。三种树脂的吸附动力学行为符合二级吸附速率方程,吸附等温线可用Langmuir等温吸附方程描述。在相同条件下,D-1300树脂的吸附量高于另外两种,因此选择安徽三星D-1300树脂作为牡丹花色苷纯化的树脂。花色苷在D-1300树脂上的穿透曲线实验数据可用Bohart-Adams模型方程拟合,拟合公式可用于给定操作条件下花色苷穿透时间的计算。牡丹花色苷提取液经过D-1300树脂动态吸附分离的适宜上样浓度为0.141mg·mL-1 ,上样流速为2BV·h-1;洗脱溶剂为60%乙醇,洗脱流速为2BV·h-1。上述操作条件下,花色苷纯度可由0.31%提高到了20.05%。4.测定了48个品种牡丹花瓣提取液的DPPH·清除活性和还原力,研究了‘洛阳红’品种的花色苷部分纯化样品清除DPPH·活性和还原力,并采用SDS-聚丙烯胺凝胶电泳法研究了其对牛血清白蛋白氧化损伤的抑制作用。结果表明:不同品种中原牡丹花色苷提取液的DPPH·清除率和还原力差异显著,分别为8.45~33.46、19.73~74.86 mg Vc/100g花瓣鲜重,且DPPH·清除率和还原力之间具有良好的线性相关关系(R2=0.9387)。‘洛阳红’部分纯化的花色苷样品对DPPH·的清除活性高于芦丁,低于抗坏血酸,半清除浓度为96.28μg·mL-1;其还原力强于芦丁,与抗坏血酸接近。0.1~500μg·mL-1花色苷样品对牛血清白蛋白(BSA)的氧化损伤具有一定抑制作用,量效关系明显。高浓度抗坏血酸和芦丁对牛血清白蛋白氧化有一定的促进作用。更多还原

【Abstract】 Tree peony is well-known in China, be named as“Wan Hua Yi Pin”and enjoy the honor of“The King of Flowers”. Flower color is one of the most important ornamental characters of tree peony . Anthocyanidins is material basis of forming flower colors of tree peony and have potential practical values bacause of many physiological activeties. The flower colors, anthocyanins, the relationship between them of Zhongyuan tree peony cultivar-groups were studied.The optimum conditions of the purification of anthocyanins from tree poney flowers were determined by the method of macroporous adsorbtion resins and the antioxidant activity of extract and anthocyanins from tree peony flower were assayed. The results were as follows:1.The flower colors of 94 cultivars of Zhongyuan tree peony were measured by a NF333 spectrophotometer based on the International Commission on Illumination(CIE) color system. CIE color system L*, a* and b* were cluster analysed by CLUSTER process using Ward square sum of deviations. The results indicated that Zhongyuan tree peony was classified into nine color series ,such as , white(20), green(1), pale red and blue(8), pale red(13), pale blue(12), red(14), purple(11), reddish purple(7) and reddish black(8). And it showed the features of the values of L*, a* and b* of different color tree peony. The relationship between L* and a*, and L* and C* were showed obviously negative correlation of white, light pink, pale red, red and pale blue, purple , reddish purple tree peony ( the correlation coefficient between L* and a* were 0.9703 and 0.6067, L* and C* were 0.8519 and 0.714). Most flower color tree peony( except red and reddish black) had notebility positive correlation between L* and b* (R2 0.8653).2. The total content and varieties of anthocyanins and the relative abundance of anthocyanin monomers in the petals of 48 cultivars of Zhongyuan tree peony were analysed by HPLC-DAD-ESI-MS. Five anthocyanins extracted from Zhongyuan tree peony were respectively cyanidin-3,5-di-O-glucoside(Cy3G5G), cyanidin-3-O-glucoside(Cy3G5G), peonidin-3,5-di-O-glucoside(Pn3G5G), peonidin-3-O- glucoside(Pn3G) and pelargonidin-3,5-di-O-glucoside(Pg3G5G), except for pelargonidin-3-O-glucoside(Pg3G). The variation range of total content of anthocyanins in 48 cultivars tree peony was 0~289.5mg/100g. The diversity of relative abundance of anthocyanin monomers in different color tree peony was obvious, and the major anthocyanins in pale red and red cultivars were Pg3G5G (respectively, 88.09 and 79.06%), belongs to PgPn and in pale blue, purple, reddish purple and reddish black cultivars Pn3G5G was major (respectively, 88.35、82.19 and 75.65、72.74%), belongs to PnCy. The relative abundance of cyanidin was bigger in reddish purple and reddish black cultivars. The relationship between flower colors and anthocyanins of tree peony was analysed, which indicated that the bright value(L*) and total anthocyanins(TA) had obviously negative correlation, and no correletion in PnCy type between a* and TA, or b* and TA but in PnPg tree peony between a* and TA displayed positive correlation, and no relation between b* and TA.3. The static adsorption characters of a variety of macroporous adsorption resins were studied, a resin fitted to purify peony anthocyanins was selected and the process parameters of Fixed-bed adsorption and desorption were determined. The results indicated that the adsorptive capacity and the rate of desorption of 3 resins D-101, D-401 and D-1300 were higher than others. The adsorption kinetics behavior was accord with the secondary adsorption rate equation and the adsorption isotherm curves could be described by Langmuir equation of the three macroporous resins. D-1300 was chosen to purify peony anthocyanin because at the same conditions the saturated adsorption amount was higher than other 2 resins. The breakthrough curve experiment data of D-1300 on anthocyanin was fitted by the Bohart-Adams equation, and the fitted formula can calculate breakthrough time of anthocyanins in given conditions. The optimum conditions of dynamic adsorption by D-1300 were as follows: the sample concentration, 0.141mg·mL-1; flow velocity, 2BV·h-1; eluent solvent, 60% ethanol; eluent velocity, 2BV·h-1. The purity of anthocyanins was increased from 0.31% to 20.05% at the conditions mentioned above.4. Anthocyanins from 48 cultivars of tree penoy flowers were extracted with methanol. Antioxidant activities were determined by the ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH·) methods. The results showed all clutivar extracts exhibited different DDPH·scavenging capacity and the reducing power, however, varying within a wide range, respectively 8.45~33.46、19.73~74.86 mg Vc/100g Petal fresh weight. Reducing power was highly correlated with DDPH·scavenging capacity (R2=0.9396). Antioxidant activities of partial puried ‘Luo Yang Hong’anthocyanin (LYHA) were further evaluated. It is found that DDPH·scavenging capacity of LYHA (IC50=96.28μg·mL-1) was higher than rutin and significantly lower than ascorbic acid, while its reduing power was much greater than rutin and equal to ascorbic acid. Also, LYHA at 0.1~500μg·mL-1 inhibited oxidative damage of Bovine Serum Albumin(BSA) induced by hydroxy radical in a concentration-dependent manner. However, rutin and ascorbic acid showed prooxidant activity towards BSA at high concentrations.更多还原