摘要
缬草为败酱科缬草属Valeriana植物,多年生草本,具有显著的平滑肌解痉、增加冠脉流量、抗心律失常、抗肿瘤、镇静安神等作用,然而对缬草的研究多集中于功效研究及化学成分分析方面,尚未有对缬草中环烯醚萜类化合物进行提取工艺优化的研究。本文以宽叶缬草V. officinalis var. latifolia为原料,探讨缬草中环烯醚萜类化合物超声波萃取工艺和大孔树脂纯化缬草中环烯醚萜的最优工艺,通过体外抗氧化活性评价试验,研究探索缬草环烯醚萜提取物清除自由基活性。研究结果如下：
(1)宽叶缬草中环烯醚萜类化合物提取工艺优化通过设计以甲醇浓度、超声波功率、萃取时间、液料比为参试因子二次通用旋转组合试验,得到超声波提取的数学模型方程：Y=1.63814+0.03575X1-0.02958X2-0.00958X3+0.02350X4-0.03433X12-0.04620X22-0.03920X32-0.03295X42-0.01000X1X3+0.01187X2X3,并且通过数学统计分析确定了宽叶缬草中提取环烯醚萜化合物最佳工艺条件：即浓度80%,超声功率250 w,萃取时间5.5 min,液料比22：1,使其理论得率为1.66%。通过试验验证理论模型可靠,验证试验值为1.66%。
(2)宽叶缬草中环烯醚萜类化合物大孔树脂富集工艺优化通过大孔吸附树脂型号优选试验、D101大孔树脂吸附环烯醚萜类化合物的上样量试验与动态洗脱试验,确定D101树脂吸附上样浓度为10.0 mg/mL,上样液的pH-6.8,上样体积为20.0 mL,洗脱体积为4 VB。通过二次通用旋转组合试验设计柱层析试验,得到D101大孔吸附树脂柱层析纯化环烯醚萜类化合物的数学模型：Y=70.06392-3.69212X1-1.84568X2+6.43739X3-2.67725X12-3.16338X22-7.88863X32+2.35000X1X3-1.57000X2X3。经过回归分析以及方差分析,对方程进行寻优环D101大孔树脂纯化烯醚萜化合物最佳工艺条件：上样流速2.5 BV/h、洗脱流速1.7 BV/h和甲醇浓度75%,其纯化后环烯醚萜纯化物的得率为72.40%,通过试验验证理论模型可靠,验证试验值为72.12%。经HPLC检测环烯醚萜纯化物纯度可以达到46.70%。
(3)缬草中环烯醚萜类化合物硅胶柱层析通过薄层层析和硅胶柱层析以及HPLC纯度检测试验研究得到氯仿：甲醇=7：1为常压硅胶柱层析的洗脱溶剂比,对目标产物即可达到很好的分离,经一次硅胶柱层析可以使经大孔吸附树脂纯化的环烯醚萜纯化物的纯度从46.70%提高到98%以上。由缬草三脂降解特征反应及HPLC检测技术,可判定纯化产物为缬草三脂。
(4)缬草中环烯醚萜类化合物纯化物抗氧化研究缬草中环烯醚萜纯化物清除自由基试验显示,缬草中环烯醚萜纯化物对于DPPH自由基、超氧阴离子自由基、羟自由基等自由基都表现出较好的清除作用,清除效果依次为DPPH·>O2·>·OH。半清除率浓度(IC50)分别为1.33、1.45、2.07μg/mL
Valeriana belongs to Valeriana L. in Valerianaceae. It has significantly effects on:smooth muscle antispasmodic, increase coronary blood flow, antiarrhythmic, anti-tumor, tranquilizing and so on. Currently, the study of effectiveness and ehemical composition analysis bout Valeriana is popular, but the study of process optimization rarely. The natural active substances of iridoids from the V. officinalis var. latifolia was extracted by the ultrasonic extraction, separated by the macroporous resin purification and silica gel column chromatography. We design experiments for discussing antioxidation in vitro of iridoids purified by macroporous resin from V. officinalis var. latifolia. Especially the process conditions. The results of study were as follows:
(1) The optimum conditions of ultrasonic extraction of iridoids form V. officinalis var. latifolia Through the quadratic general rotary unitized design, ultrasonic extraction mathematical simulation model was concluded by the linear regression analysis:Y=1.63814+0.03575X1-0.02958X2-0.00958X3+0.02350X4-0.03433X12-0.04620X22-0.03920X32-0.03295X42-0.01000X1X3+0.01187X2X3 Then the optimum conditions of ultrasonic extract iridoids form V. officinalis var. latifolia was determinded by the mathematical statistical analysis:ethanol concentration is 80%, ultrasonic power is 250 w, extract time is 5.5 min, ratio of material to liquid is 22:1. Its extraction rate on theory which was calculated by mathematical simulation model can be achieved 1.66%. The theoretical model was proved that was reliable by the verification experiments, the value is 1.66%.
(2) The process optimization of iridoids purified by macroporous resin After the type optimized test of macroporous adoption resin, D101 resin the loading volume test and the dynamic eluting test and the column chromatography quadratic general rotary experiments on three factors (loading velocity, eluting velocity and concen-tration of eluting solvent). the optimum conditions of D101 macroporous resin column chromatography test was determined by these experiments and all kinds of analysis: the concentration of sample is 10.0 mg/mL, pH=6.8, the upper sample volume is 20 mL, elution volume is 4 VB, macroporous resin purification mathematical model of iridoids can be achieved:Y=70.06392-3.69212X1- 1.84568X2+6.43739X3-2.67725X12-3.16338X22-7.88863X32+2.35000X1X3-1.57000X2X3, Through regression analysis and variance analysis, the optimum conditions on macroporous resin purification were drawn:the sample velocity is 2.5 BV/h, the elution flow is 1.7 BV/h, the ethanol concentration is 75%, the yield rate of purified iridoids is 72.40%, which was measured by HPLC, and the purity of separated iridoids can reach 46.70%.
(3) The purification of iridoids by silica gel column chromatography Through the TLC, silica gel column chromatography and HPLC purity experiments, chloroform and methanol in the ratio of 7:1 as the eluting solvent, used the ratio can be achieve good separated effect to target product. With the silica gel column chromatography the purity of purified iridoids powder by the macroporous adoption resin can be advanced from 46.70% to 98%. The purificated products were valtrale Judged form characteristics reaction and HPLC detect technology.
(4) Antioxidantion in vitro research of the iridoids purificated by macroporous resin In the experiment of scavenging on free radical invitro, the purification of iridoids showed the scavenging effect on DPPH, hydroxyl radical(-OH), superoxide anion radical (O2·) and the scavenging order was DPPH>O2·>·OH, the half scaven-ging concentration (IC50) were 1.33,1.45,2.07μg/mL, respectively.
引文
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