匀浆提取银中杨酚苷及其体外抗PRV活性与清除DPPH自由基能力的研究
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摘要
本文在建立总酚苷可见分光光度法检测方法和水杨苷含量高效液相色谱法的检测方法的基础上,进行银中杨中酚苷匀浆提取工艺的影响因素试验研究。在单因素试验的基础上,应用响应面法优化工艺,得到最佳工艺和银中杨酚苷提取物,并研究了银中杨酚苷提取物大孔吸附树脂纯化的方法。在对猪伪狂犬病病毒(SC毒株)的分离及鉴定的基础上,完成银中杨酚苷体外抗伪狂犬病病毒(PRV)的活性试验,并进行了银中杨酚苷与PRV相互作用荧光定量PCR检测。同时,通过清除DPPH能力测试方法考查了银中杨酚苷的抗氧化能力。具体研究结果如下：
1、采用响应面法对匀浆提取银中杨干树皮、新鲜树皮、新鲜芽鳞、鲜叶、新鲜芽鳞和枝叶混合物(W芽鳞：W枝叶=1：1)五种原料中总酚苷、水杨苷成分进行优化。考察了匀浆时间、乙醇体积分数、匀浆次数、料液比、粒径大小等因素对提取工艺的影响,应用了Box-Behnken的实验设计,选取总酚苷、水杨苷纯度与得率作为响应值。响应面法优化后提取工艺条件为：
银中杨干树皮酚苷提取条件：提取时间5min、乙醇体积分数10%、液料比10：1mL/g、提取次数1次。在所确定的工艺条件下,酚苷纯度3.654%；水杨苷纯度0.3726%；得率6.5571%；银中杨新鲜树皮酚苷提取条件：提取时间5min、乙醇体积分数15%、液料比5：1mL/g、提取次数1次。在所确定的工艺条件下,酚苷纯度2.4289%;水杨苷纯度0.3099%；得率2.5786%；银中杨新鲜芽鳞酚苷提取条件：提取时间3mmin、乙醇体积分数10%、液料比15：1mL/g、提取次数1次。在所确定的工艺条件下,酚苷纯度2.0615%；水杨苷纯度0.7396%；得率4.877%；银中杨鲜叶酚苷提取条件：提取时间3mmin、乙醇体积分数15%、液料比10：1mL/g、提取次数1次。在所确定的工艺条件下,酚苷纯度5.554%；水杨苷纯度0.5049%；得率4.4156%；银中杨新鲜芽鳞和枝叶混合物酚苷提取条件：提取时间5min、乙醇体积分数20%、液料比10：1mL/g、提取次数1次。在所确定的工艺条件下,酚苷纯度4.2021%；水杨苷纯度1.0679%；得率4.8915%。
经过HPD-700大孔树脂吸附实验,酚苷、水杨苷含量分别从44.0561%和4.0616%提高到86.4466%和9.1899%,回收率80.2341%。
2、银中杨酚苷体外具有抗PRV感染细胞的能力,通过观察细胞病变效应(CPE),在同等条件下,鲜树皮酚苷提取物体外抗PRV效果最好,其次为干树皮酚苷提取物,都优于利巴韦林和水杨苷。对107TCIDso PRV,半数有效抑制浓度分别为：干树皮酚苷提取物IC50577.981ug/ml、鲜树皮酚苷提取物IC50561.35ug/ml。
根据PRV-SC毒株的gE基因参考序列,建立PRV荧光定量PCR方法：构建阳性标准品(PRV-gE质粒),进行PCR鉴定。经序列测定,测定结果与参考毒株的基因序列完全一致。建立荧光定量PCR标准曲线,进行敏感性试验和特异性试验：得到标准曲线方程为Y=3.489*LOG(X)+46.81,相关系数R2=0.999,扩增效率E=0.935。对银中杨酚苷与PRV相互作用后,PRV载量进行定量分析。定量PCR拷贝数与CPE结果相符,PRV载量有明显的变化。
3、对DPPH的清除能力以半清除率浓度ICs0表示,各抗氧化剂的抗氧化能力的大小顺序为银中杨新鲜枝叶芽鳞混合物酚苷>银中杨新鲜鳞芽酚苷>VC>银中杨鲜叶酚苷>银中杨鲜皮酚苷>银中杨干皮酚苷>BHT。ICso值分别为0.3519mg/mL、0.3553mg/mL、0.3742mg/mL、0.3810mg/mL、0.4492mg/mL、0.5288mg/mL、0.6277mg/mL。
银中杨清除DPPH·的能力与酚苷含量成正相关,银中杨新鲜枝叶芽鳞混合物酚苷、银中杨新鲜鳞芽酚苷清除DPPH·的能力均强VC,各酚苷清除DPPH·的能力均优于BHT,其中银中杨新鲜枝叶芽鳞混合物的酚苷提取物清除DPPH·的能力最强。
本研究的主要创新性表现在三个方面：首次应用匀浆提取的方法从银中杨中提取酚苷；首次验证银中杨酚苷具有体外抗PRV病毒活性作用；首次验证银中杨酚苷提取物具有清除DPPH自由基的能力,新鲜原料提取物的这种能力体现更为明显。
本研究的目的意义在于在自主创新的生态工艺理论体系指导下,使用匀浆提取的生态分离方法完成银中杨酚苷的提取分离,得到银中杨酚苷提取物,对银中杨的资源实现最大化的利用,最终以尽可能少的资源消耗和尽可能小的环境代价实现最大的经济效益和生态效益。
In this study,UV/Visible spectrophotometer and high performance liquid chromatography (HPLC)detection methods has been established for the determination of the total phenolic glycosides(PGs) and salicin.Research on impact factors on the basis of single factor tesin the PGs homogenates extraction process.Using response surface methodology (RSM) to get the optimizing PGs extract process.Extracting and purifying with macroporous resins was found to be an efficient potential method for PGs extracts.Pseudorabies virus (PRV),a swine neurotropic alphaherpesvirus, was separated and identified as PRV-SC strain in this paper. An in vitro model was used to examine anti-PRV activity test of PGs.Fluorescence Quantitative PCR in detection of PGs-PRV interaction. At the same time, the ability to clear the DPPH test methods to examine PGs antioxidant capacity. The findings are as follows:
First, using the response surface method of dry barks, fresh bark, fresh buds, fresh leaves, and fresh buds,leaves mixture (1:1) five kinds of raw materials to test ando ptimize total PGs, salicin component. The homogenate time, ethanol volume fraction, the homogenate number, solid-liquid ratio, particle size and other factors on the extraction process, the application of the Box-Behnken experimental design, select the concentration and yield of total PGs, salicin as a response value.
In the optimized model,several response plots were generated to examine parameter effects on PGs RSM extract process:
Dry barks PGs extraction conditions:5min extraction time,10%ethanol concentration, Solid-liquid ratio10:1mL/g, one extraction times. Determined by process conditions, the total PGs concentration is3.654%; salicin concentration is0.3727%; yield is6.5571%; fresh barks PGs extraction conditions:5min extraction time,15%ethanol concentration, Solid-liquid ratio5:1mL/g, one extraction times. Determined by process conditions, the total PGs concentration is2.4289%,salicin concentration is0.3099%,yield is2.5786%;Fresh buds PGs extraction conditions:extraction time3min,10%ethanol concentration, Solid-liquid ratio15:1mL/g, one extraction times, Determined by process conditions, the total PGs concentration is2.0615%, salicin concentration is0.7396%; yield of4.877%; Fresh leaves PGs extraction conditions:3min extraction time,15%ethanol concentration. Solid-liquid ratio10:1mL/g, one extraction times.Determined by process conditions, the total PGs concentration of5.554%; salicin concentration is0.5049%,yield is4.4156%; Mixture of fresh buds and leaves PGs extraction conditions:5min extraction time,20%ethanol concentration, Solid-liquid ratio10:1mL/g, one extraction times. Determined under the process conditions, the total PGs concentration is4.2021%, salicin concentration is1.0679%purity; yield is4.8915%.
After macroporous resin adsorption and desorption experiments, PGs, salicin content increased from44.0561%and4.0616%to86.4466%and9.1899%,80.2341%recovery by HPD-700.
Second, PGs in vitro anti-PRV-infected cells, by observing the cytopathic effect (CPE), under the same conditions, the fresh bark PGs extracts anti-PRV results is the best, followed by dry barks PGs extract.They are superior to ribavirin and salicin. Median effective inhibitory concentration107TCID50PRV:Dry barks PGs extract IC50is1147.981ug/ml, the fresh barks PGs extract IC50is561.3489ug/ml.
The gE gene reference sequence according to the strain of PRV-SC, PRV fluorescent quantitative PCR method:build a positive standard (PRV-gE plasmid), identified by PCR. Determination of the sequence, the measurement results with the gene sequence of the reference strain completely consistent, quantitative PCR standard curve, sensitivity test and specificity of the test:Standard curve equation Y=3.489*LOG(X)+46.81, R2=0.999, amplification efficiency E=0.935.
Interaction PGs-PRV,PRV load for quantitative analysis. Quantitative PCR copy number is consistent with the CPE results:PGs-PRV interaction, under the same conditions, fresh bark PGs extract is the best, followed by fresh bark PGs extract, They are superior to ribavirin and salicin, PRV significant changes in the load.
Third, DPPH scavenging half-clearance concentration IC50, the size of the order of the antioxidant capacity of antioxidants:Mixture of fresh buds and leaves PGs extract> fresh buds PGs extract> VC> fresh leaves PGs extract> BHA> fresh barks PGs extract> dry barks PGs extract. IC50values were0.3519mg/mL,0.3553mg/mL,0.3742mg/mL,0.3810mg/mL,0.4492mg/mL,0.5288mg/mL,0.6277mg/mL.
Populus clear DPPH capacity and PGs content as being relevant,the capacity of Mixture of fresh buds and leaves PGs extract and fresh buds PGs extract is strong than VC; the various PGs clear DPPH the ability are superior to BHT. Mixture of fresh buds and leaves PGs extract Clearing DPPH is strongest.
The major innovation of this study mainly in three aspects:First, application homogenates extraction process to get PGs extract s from Populus parts. Second, First validation of Populus PGs with in vitro anti-PRV virus activity. Third,The PGs extracts fresh ingredients have clear DPPH capacity antioxidant activity.
The purpose and the significance of this study:Under the guidance of the theory of the ecological process of independent innovation system to complete the homogenates extraction and separation of PGs. and ultimately to the minimal resource consumption and environmental costs as much as possible to maximize the economic and ecological benefits.
1、采用响应面法对匀浆提取银中杨干树皮、新鲜树皮、新鲜芽鳞、鲜叶、新鲜芽鳞和枝叶混合物(W芽鳞：W枝叶=1：1)五种原料中总酚苷、水杨苷成分进行优化。考察了匀浆时间、乙醇体积分数、匀浆次数、料液比、粒径大小等因素对提取工艺的影响,应用了Box-Behnken的实验设计,选取总酚苷、水杨苷纯度与得率作为响应值。响应面法优化后提取工艺条件为：
银中杨干树皮酚苷提取条件：提取时间5min、乙醇体积分数10%、液料比10：1mL/g、提取次数1次。在所确定的工艺条件下,酚苷纯度3.654%；水杨苷纯度0.3726%；得率6.5571%；银中杨新鲜树皮酚苷提取条件：提取时间5min、乙醇体积分数15%、液料比5：1mL/g、提取次数1次。在所确定的工艺条件下,酚苷纯度2.4289%;水杨苷纯度0.3099%；得率2.5786%；银中杨新鲜芽鳞酚苷提取条件：提取时间3mmin、乙醇体积分数10%、液料比15：1mL/g、提取次数1次。在所确定的工艺条件下,酚苷纯度2.0615%；水杨苷纯度0.7396%；得率4.877%；银中杨鲜叶酚苷提取条件：提取时间3mmin、乙醇体积分数15%、液料比10：1mL/g、提取次数1次。在所确定的工艺条件下,酚苷纯度5.554%；水杨苷纯度0.5049%；得率4.4156%；银中杨新鲜芽鳞和枝叶混合物酚苷提取条件：提取时间5min、乙醇体积分数20%、液料比10：1mL/g、提取次数1次。在所确定的工艺条件下,酚苷纯度4.2021%；水杨苷纯度1.0679%；得率4.8915%。
经过HPD-700大孔树脂吸附实验,酚苷、水杨苷含量分别从44.0561%和4.0616%提高到86.4466%和9.1899%,回收率80.2341%。
2、银中杨酚苷体外具有抗PRV感染细胞的能力,通过观察细胞病变效应(CPE),在同等条件下,鲜树皮酚苷提取物体外抗PRV效果最好,其次为干树皮酚苷提取物,都优于利巴韦林和水杨苷。对107TCIDso PRV,半数有效抑制浓度分别为：干树皮酚苷提取物IC50577.981ug/ml、鲜树皮酚苷提取物IC50561.35ug/ml。
根据PRV-SC毒株的gE基因参考序列,建立PRV荧光定量PCR方法：构建阳性标准品(PRV-gE质粒),进行PCR鉴定。经序列测定,测定结果与参考毒株的基因序列完全一致。建立荧光定量PCR标准曲线,进行敏感性试验和特异性试验：得到标准曲线方程为Y=3.489*LOG(X)+46.81,相关系数R2=0.999,扩增效率E=0.935。对银中杨酚苷与PRV相互作用后,PRV载量进行定量分析。定量PCR拷贝数与CPE结果相符,PRV载量有明显的变化。
3、对DPPH的清除能力以半清除率浓度ICs0表示,各抗氧化剂的抗氧化能力的大小顺序为银中杨新鲜枝叶芽鳞混合物酚苷>银中杨新鲜鳞芽酚苷>VC>银中杨鲜叶酚苷>银中杨鲜皮酚苷>银中杨干皮酚苷>BHT。ICso值分别为0.3519mg/mL、0.3553mg/mL、0.3742mg/mL、0.3810mg/mL、0.4492mg/mL、0.5288mg/mL、0.6277mg/mL。
银中杨清除DPPH·的能力与酚苷含量成正相关,银中杨新鲜枝叶芽鳞混合物酚苷、银中杨新鲜鳞芽酚苷清除DPPH·的能力均强VC,各酚苷清除DPPH·的能力均优于BHT,其中银中杨新鲜枝叶芽鳞混合物的酚苷提取物清除DPPH·的能力最强。
本研究的主要创新性表现在三个方面：首次应用匀浆提取的方法从银中杨中提取酚苷；首次验证银中杨酚苷具有体外抗PRV病毒活性作用；首次验证银中杨酚苷提取物具有清除DPPH自由基的能力,新鲜原料提取物的这种能力体现更为明显。
本研究的目的意义在于在自主创新的生态工艺理论体系指导下,使用匀浆提取的生态分离方法完成银中杨酚苷的提取分离,得到银中杨酚苷提取物,对银中杨的资源实现最大化的利用,最终以尽可能少的资源消耗和尽可能小的环境代价实现最大的经济效益和生态效益。
In this study,UV/Visible spectrophotometer and high performance liquid chromatography (HPLC)detection methods has been established for the determination of the total phenolic glycosides(PGs) and salicin.Research on impact factors on the basis of single factor tesin the PGs homogenates extraction process.Using response surface methodology (RSM) to get the optimizing PGs extract process.Extracting and purifying with macroporous resins was found to be an efficient potential method for PGs extracts.Pseudorabies virus (PRV),a swine neurotropic alphaherpesvirus, was separated and identified as PRV-SC strain in this paper. An in vitro model was used to examine anti-PRV activity test of PGs.Fluorescence Quantitative PCR in detection of PGs-PRV interaction. At the same time, the ability to clear the DPPH test methods to examine PGs antioxidant capacity. The findings are as follows:
First, using the response surface method of dry barks, fresh bark, fresh buds, fresh leaves, and fresh buds,leaves mixture (1:1) five kinds of raw materials to test ando ptimize total PGs, salicin component. The homogenate time, ethanol volume fraction, the homogenate number, solid-liquid ratio, particle size and other factors on the extraction process, the application of the Box-Behnken experimental design, select the concentration and yield of total PGs, salicin as a response value.
In the optimized model,several response plots were generated to examine parameter effects on PGs RSM extract process:
Dry barks PGs extraction conditions:5min extraction time,10%ethanol concentration, Solid-liquid ratio10:1mL/g, one extraction times. Determined by process conditions, the total PGs concentration is3.654%; salicin concentration is0.3727%; yield is6.5571%; fresh barks PGs extraction conditions:5min extraction time,15%ethanol concentration, Solid-liquid ratio5:1mL/g, one extraction times. Determined by process conditions, the total PGs concentration is2.4289%,salicin concentration is0.3099%,yield is2.5786%;Fresh buds PGs extraction conditions:extraction time3min,10%ethanol concentration, Solid-liquid ratio15:1mL/g, one extraction times, Determined by process conditions, the total PGs concentration is2.0615%, salicin concentration is0.7396%; yield of4.877%; Fresh leaves PGs extraction conditions:3min extraction time,15%ethanol concentration. Solid-liquid ratio10:1mL/g, one extraction times.Determined by process conditions, the total PGs concentration of5.554%; salicin concentration is0.5049%,yield is4.4156%; Mixture of fresh buds and leaves PGs extraction conditions:5min extraction time,20%ethanol concentration, Solid-liquid ratio10:1mL/g, one extraction times. Determined under the process conditions, the total PGs concentration is4.2021%, salicin concentration is1.0679%purity; yield is4.8915%.
After macroporous resin adsorption and desorption experiments, PGs, salicin content increased from44.0561%and4.0616%to86.4466%and9.1899%,80.2341%recovery by HPD-700.
Second, PGs in vitro anti-PRV-infected cells, by observing the cytopathic effect (CPE), under the same conditions, the fresh bark PGs extracts anti-PRV results is the best, followed by dry barks PGs extract.They are superior to ribavirin and salicin. Median effective inhibitory concentration107TCID50PRV:Dry barks PGs extract IC50is1147.981ug/ml, the fresh barks PGs extract IC50is561.3489ug/ml.
The gE gene reference sequence according to the strain of PRV-SC, PRV fluorescent quantitative PCR method:build a positive standard (PRV-gE plasmid), identified by PCR. Determination of the sequence, the measurement results with the gene sequence of the reference strain completely consistent, quantitative PCR standard curve, sensitivity test and specificity of the test:Standard curve equation Y=3.489*LOG(X)+46.81, R2=0.999, amplification efficiency E=0.935.
Interaction PGs-PRV,PRV load for quantitative analysis. Quantitative PCR copy number is consistent with the CPE results:PGs-PRV interaction, under the same conditions, fresh bark PGs extract is the best, followed by fresh bark PGs extract, They are superior to ribavirin and salicin, PRV significant changes in the load.
Third, DPPH scavenging half-clearance concentration IC50, the size of the order of the antioxidant capacity of antioxidants:Mixture of fresh buds and leaves PGs extract> fresh buds PGs extract> VC> fresh leaves PGs extract> BHA> fresh barks PGs extract> dry barks PGs extract. IC50values were0.3519mg/mL,0.3553mg/mL,0.3742mg/mL,0.3810mg/mL,0.4492mg/mL,0.5288mg/mL,0.6277mg/mL.
Populus clear DPPH capacity and PGs content as being relevant,the capacity of Mixture of fresh buds and leaves PGs extract and fresh buds PGs extract is strong than VC; the various PGs clear DPPH the ability are superior to BHT. Mixture of fresh buds and leaves PGs extract Clearing DPPH is strongest.
The major innovation of this study mainly in three aspects:First, application homogenates extraction process to get PGs extract s from Populus parts. Second, First validation of Populus PGs with in vitro anti-PRV virus activity. Third,The PGs extracts fresh ingredients have clear DPPH capacity antioxidant activity.
The purpose and the significance of this study:Under the guidance of the theory of the ecological process of independent innovation system to complete the homogenates extraction and separation of PGs. and ultimately to the minimal resource consumption and environmental costs as much as possible to maximize the economic and ecological benefits.
引文
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