In-Vitro Anti-PRV Activity And DPPH·radical Scavenging Activity Of Phenolic Glycosides Homogenated Extraction From Popolus Alba×P. Berolinensis

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.