Studies On The Extraction Process And Antioxidant Mechanism Of Taxifolin From Wood Sawdust Of Larix Gmelinii(Rupr.) Rupr

In this study,the extraction methods of powerful antioxidant taxifolin and antioxidant mechanism of taxifolin in Larix gmelinii(Rupr.)Rupr wood sawdust were studied.Finally,a set of process which employed enzyme-induced ultrasonic extraction,macroporous resins enrichment as well as column chromatography puritification was established for the extraction and purification of taxifolin.Meanwhile,the antioxidant activities of taxifolin about Nrf2 signaling pathway were introduced for the first time.This study for the research and development of forestry biological agents provides a new idea.The results are as follow:1.A HPLC method for simultaneous determination of taxifolin was developed as follows:Chromatographic column HIQ sil C18V(250 mm×4.6 mm I.D.),mobile phase methanol-water-acetic acid(44:56;0.4,v/v/v),flow rate 1 mL/min,injection volume 10 ?L,column temperature 35?,UV at 290 nm.A good linearity was achieved for taxifolin in concentration ranges of 0.30-300 ?g/mL.The developed method was validated with satisfactory repeatability and precision(RSD<3.0%).2.Effects of ultrasound extraction method on the contents of taxifolin in Larix gmelini(Rupr.)Rupr wood sawdust were investigated.The optimum process of ultrasound extraction method was confirmed for the first time,and the main parameters affecting the contents were optimized as follows:Extraction method:ultrasound extractionExtraction liquid:80%ethanolLiquid to solid ratio:20:1Extaction time:2.5 minExtaction temperature:60?Extaction times:3 times3.Effects of enzyme incubation-water extraction(El-WE)method on the contents of taxifolin in Larix gmelini(Rupr.)Rupr wood sawdust were investigated.The optimum process of nzyme incubation-water extraction(El-WE)method was confirmed for the first time,and the main parameters affecting the contents were optimized as follows:Enzyme induction:enzyme-assisted ultrasonic extractionEnzyme type:cellulose and pectinaseConcentration of enzymes:0.5 mg/ml cellulase and 0.5 mg/ml pectinaseInduction time:18 hInduction temperature:30?pH:5.0After enzyme induction treatment,the yields of taxifolin and total flavonoids increased from 1.06±0.08 to 1.35±0.04 mg/g and 4.13±0.17 to 4.96±0.29 mg/g,respectively.4.The separation of taxifolin by macroporous resina was studied for the first time and the optimum adsorption and desorption parameters on the optimal AB-8 resin were obtained.Sample concentration:30 mg/mLSample volume:3 mLAdsorption flow rate:0.4 mL/minDesorption solution and volume:ethanol-water(10:90,v/v)solution 5 BV and followed by ethanol-water(20:80,v/v)solution 25 BVDesorption flow rate:1 mL/minAfter treated by AB-8 resin under the optimal conditions,the contents of taxifolin were increased from 1.54%to 43.31%.The recovery yield was 90.02%.5.The purification of taxifolin by silica gel was studied for the first time.Normal phase silica gel was chosen as filter.Chloroform-methanol system was established as desorption solution.Sample to silica gel ratio:1:15Desorption solution and volume:chloroform 100%,chloroform-methanol(30:1,V/V),chloroform-methanol(25:1,V/V),chloroform-methanol(20:1,V/V)Taxifolin needle-like crystals was obtained in the elution part of the chloroform-methanol(20:1,V/V).The contents of taxifolin were 94.35%.The recovery yield was 72.46%.6.Antioxidant activity assessment and cell oxidation mechanism research showed that taxifolin had strong antioxidant activities.Taxifolin exerts cytoprotective effects against oxidative stress through the Nrf2-dependent antioxidant pathway,which was studied for the first time.DPPH radical scavenging,reducing power assay and DNA damage protection assay were used to evaluated the antioxidant properties of taxifolin.The IC50 value of taxifolin were 22.57±0.24 and 9.06±0.19 by DPPH radical scavenging,reducing power assay,respectively.Furthermore,XOD,SOD and GR activities was tested in HepG2 cells to evaluate effects of enzyme system.The results showed taxifolin could inhibit XOD activity,enhance SOD and GR activities in HepG2 cell with in a concentration dependment manner.Our results indicated taxifolin treatment enhanced the phosphorylation-dependent activation of signaling components,such as PI3K/AKT,ERK,and JNK.Meanwhile,taxifolin can induce Nrf2-dependent antioxidant response element(ARE)activity and gene expression of heme oxygenase-1(HO-1)and NAD(P)H quinone oxidoreductase 1(NQO1).The experimental results will provide useful scientific reference for the natural resource protection of Larix gmelinii(Rupr.)Rupr.