急性子多糖的含量测定

1、急性子多糖的含量测定目 录摘 要21 材料与方法32 结果与讨论33 小结与讨论6参 考 文 献6致 谢 6急性子多糖的含量测定 摘 要摘要：目的：利用响应面分析法对急性子多糖的提取工艺进行优化。方法：以提取温度、提取时间、料液比为自变量，多糖得率为因变量，通过对自变量各水平的多元线性回归及二项式拟合，用星点设计-响应面法选取最佳工艺，并进行预测分析。结果：多糖得率最大值为3.73%，最优工艺条件为：料液比为1:25.8，提取温度为92.0，浸提时间为120.5min，浸提2次；提取实际值与预测值偏差为-2.9%。结论：方法简便合理，稳定，可预测性较优。 关键词： 急性子；多糖；星点设计；响应

2、面法急性子为凤仙花科植物凤仙花Impatiens balsamina L.的干燥成熟种子，始载于救荒本草，为我国传统药物之一；性温、味微苦，归肝、脾经；具有破血软坚、消积的功效，用于癓瘕痞块、经闭、噎膈1。本品除含脂肪油、挥发油外，还含多糖、蛋白质、皂苷、黄酮类等化合物，本实验采用响应面分析法对急性子多糖的提取工艺进行了研究，该实验方法应用于中药材提取工艺研究报道较少，国内常用均匀设计和正交设计进行优化，但这两种方法实验精度不够，建立的数学模型预测性较差2，其回归结果多为线性模型，而星点设计实验所得的信息量大、直观、更适合于探索性试验研究，本研究可为急性子多糖类化学成分研究提供科学参考。1 材

3、料与方法1.1 仪器与试剂 紫外可见分光光度计（UV24802型），电子天平（上海天平仪器厂）。葡萄糖、浓硫酸、苯酚、95%乙醇皆为分析纯，水为重蒸水。1.2 材料 急性子为凤仙花科植物凤仙花Impatiens balsamina L.的干燥成熟种子，采自鞍山千山，经本校中药鉴定教研室翟延君教授鉴定。1.3 实验方法1.3.1提取方法精确称取急性子药材粉末5.0 g，置250 mL圆底烧瓶中，加80乙醇100 mL加热回流1.5 h，过滤、挥干。干燥后的药材粉末用20倍量水在70水浴回流提取2 h，残渣用适量水洗涤，滤液于250 mL量瓶中定容。1.3.2标准曲线的制备精密称取干燥至恒重的葡萄

4、糖粉末26.85 mg，至于100 mL容量瓶中，加水定容至刻度，摇匀，配得葡萄糖标准溶液。精密量取葡萄糖标准溶液0、5、10、15、20、25 mL稀释至100 mL量瓶中，取各浓度的标准液1 mL分别置于试管中，并加5%苯酚溶液1.0 mL，摇匀，再加入浓硫酸5.0 mL，摇匀后，置40 水浴中加热15 min，取出，室温放置10 min，在490 nm处测定其吸光值。得回归方程C=106.5A + 3.797，相关系数r=0.9995。1.3.3急性子多糖的测定精密吸取样品溶液1 mL置于25 mL量瓶中，加水定容。精密量取1 mL，按1.3.2项显色测量。2 结果与讨论2.1 急性子多

5、糖水提过程中单因素的影响研究 2.1.1 提取温度考察 精密称取5.0 g干燥至恒重的药材粉末，按1.3.1项下方法操作，分别在40、50、60、70、80、90、100 温度下提取，测定多糖含量。随着温度的升高，多糖溶出量逐渐增大且多糖溶液颜色加深，在90提取率达到最大值。高温可能对多糖结构和活性有一定影响，所以提取温度不宜超过100 。结果见图1。2.1.2 提取时间考察 精密称取5.0 g干燥至恒重的药材粉末，按1.3.1项下方法操作，分别提取60、100、140、180、220、260 min，测定多糖含量。当提取时间低于140 min时，细胞壁的溶胀和破坏程度不够，多糖不能完全溶出，

6、提取率较低。提取时间至140 min时，多糖已基本被溶出。因此从能耗与时间等因素考虑，提取时间以140 min左右为宜。2.1.3 料液比考察 精密称取5.0 g干燥至恒重的药材粉末，按1.3.1项下方法操作，采用料液比1:5、1:10、1:15、1:20、1:25、1:30、1:35分别提取，测定多糖含量。多糖提取率随着料液比的增加而增加，1:25时提取率最高，多糖基本提取完全，增大加入溶剂比对多糖提取率影响较弱，因此选择料液比1:25左右为宜，结果见图3。2.1.4 提取次数考察 精密称取5.0 g干燥至恒重的药材粉末，按1.3.1项下方法操作，对物料连续提取4次，测定多糖含量。当提取2次

7、时提取率几乎为零，说明多糖提取完全。因此选择提取次数2次。2.2 星点设计实验2.2.1 模型拟合 综合单因素实验结果，提取因素为提取温度、提取时间和料液比，因提取次数为非连续变量，不能进行回归处理，固定提取两次，分析因素表见表1，星点设计方案及实验结果见表2。表1 因素与水平Table 1 Factors and levels因素水平-1.732-10+1+1.732X1温度/6068.58091.5100X2时间/min80105.4140174.6200X3料液/g/ml1014.22025.830表2 星点试验与结果Table 2 Results of CCD-RSM test试验号X

8、1X2X3多糖提取率(%)1-1-1-11.732+1-1-11.983-1+1-10.474+1+1-10.505-1-1-12.006+1-1+11.877-1+1+13.568+1+1+13.609-1.732001.5210+1.732002.55110-1.73201.91120+1.73202.201300-1.7320.421400+1.7322.9715200003.33将所得数据用Design-Expert 8.0.2（试用版）软件进行效应面试验分析，以多糖得率为效应值分别对各因素进行多元线性回归和二项式方程拟合。经软件对表2中试验数据分析后发现用二阶多项式回归较优，得以多糖

9、得率(Y)为效应值，3个参数(X1X2X3)为自变量的二次回归方程模型：Y=3.48+0.14X1+0.076X2+0.77X3-5.016E-003X1X2-0.045X1X3+0.75X2X3-0.48X12-0.47X22-0.59X32(R2=0.9824) 对二项式方程中的各项系数进行F检验，删除不显著因素，方程简化为：Y=3.48+0.14X1+0.076X2+0.77X3+0.75X2X3-0.48X12-0.47X22-0.59X32(R2=0.9817)由方差分析表结果可知，提取温度对多糖得率有显著影响，料液比有极显著影响，提取时间和料液比的交互作用对其有极显著影响，二项式中

10、的X2偏回归系数达极显著水平。回归方程失拟性检验P=0.07290.05,差异不显著；总回归方程PF)显著性ModelX123.220.28912.580.2872.747.730.00010.0194*X20.0810.082.240.1650X38.2318.23231.210.0001*X1X2X1X3X2X3X1X1X2X2X3X3残差失拟性纯误差总差2.013E-004 0.0164.553.583.475.480.360.290.06923.581111111055192.013-0040.0164.553.583.475.480.0360.0570.0145.652E-0030.

11、45127.80100.4897.37153.874.140.94160.51540.00010.00010.00010.00010.0729* 注：*为极显著(P0.001)，*为非常显著(P0.01)，*为显著(P0.05)。2.2.2 工艺参数优化与预测 把因变量与另两个因素拟合为三维曲面图，固定三个变量中的一个为中值，以拟合目标函数为数学模型，绘制因变量曲面图，由回归模型进行预测分析，多糖得率最大值为3.73%，最优工艺为：水为溶剂，在92.0，料液比1:25.8，提取120.5min，提取2次。根据最优工艺进行验证，实际测得率为3.62%，实际值比预测值低2.9%。3 小结与讨论3.

12、1 响应面优化的前提是设计的实验点应包括最佳的实验条件，如果实验点选取不当，就不能得到很好的优化结果。在使用响应面优化法之前，应确立合理的实验各因素与水平。因此本实验首先采用单因素考察，以确定合理的各实验条件。3.2 由于响应面优化法采用非线性数学模型拟合，在中心点进行重复性实验以提高实验的精确度，预测值更接近真实值。所以本实验采用该法对急性子的多糖提取方法进行了研究，实验方法简便合理、稳定，可预测性较优。 3.3 本实验在单因素考察基础上建立三因素五水平响应面实验方法，最终确立急性子多糖的最佳提取工艺条件为：温度为92.0，时间为120.5min，料液比为25.8:1；二项式拟合判定系数较高

13、（R2=0.9817），按此工艺急性子多糖的提取率可达3.62%，与预测值偏差-2.9%。参考文献格式如下：参考文献 1中国药典（一部）S.2010：247.2 吴伟，崔光华星点设计一效应面优化法及其在药学中的应用J国外医学药学分册，2000，27（5）：292.内部资料请勿外传9JWKffwvG#tYM*Jg&6a*CZ7H$dq8KqqfHVZFedswSyXTy#&QA9wkxFyeQ!djs#XuyUP2kNXpRWXmA&UE9aQGn8xp$R#͑GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnG

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28、T#&ksv*3tnGK8!z89AmYWpazadNuGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849

29、GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89A

30、mYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqvadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcv

31、R9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89Amv$UE

32、9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadN

33、u#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz84!z89Amv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7J

34、nD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!

35、zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%MadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&q

36、YpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*adNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGj

37、qv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89Amv$UE9wEwZ#QcUE%&qYpEh5p

38、Dx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6

39、YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz84!z89Amv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!z

40、n%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9w

41、EwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNuGK8!z89AmYWpazadNu#KN

42、&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP

43、$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz8

44、49GxGjqv3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&

45、qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89Amv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXR

46、m6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%MzadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA

47、5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYp

48、Eh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!z

49、n%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89Amv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEw

50、Z#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz84!z89Amv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEw

51、Z#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrW

52、wcvR9&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$U*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849

53、GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNuGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z8

54、9AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxGjqv$UE9wEwZ#QcUE%&qYpEh5pDx2zVkum&gTXRm6X4NGpP$vSTT#&ksv*3tnGK8!z89AmYWpazadNu#KN&MuWFA5uxY7JnD6YWRrWwcvR9CpbK!zn%Mz849GxGjqvUE%&qYpEh5pDx2zVku