Complanatus Phenolic Compounds And Antioxidant Compounds By Hplc Screening

In this dissertation, the phenolics in Semen Astragali Complanati, a native medicinal seed in Shaanxi province, were studied for its extraction, purification, antioxidant capacity, components identification, and screening of antioxidants by high performance liquid chromatography (HPLC). This systematical research was expected to provide theoretical basis and technology parameters for the development and deep-processing of Semen Astragali Complanati.Nine chapters are included in the dissertation. The contents and results are following:1. The optimum extraction conditions of phenolcs from Semen Astragali Complanati under the ultrasonic condition of 120 W and 40.0 kHz were as follows:With the.60%(v/v) methanol as extractant, the optimized conditions were extraction temperature of 42℃, extraction time of 47min, solvent-to-sample ratio,30:1 and particle size,120mesh. Under the optimized conditions, the extraction yield was 10.66%.With the 60%(v/v) ethanol as extractant, the optimized conditions were extraction temperature of 52 C, extraction time of 34min, solvent-to-sample ratio,26:1 and particle size, 100mesh. Under the optimized conditions, the extraction yield was 7.08%.With the 60% (v/v) acetone as extractant, the optimized conditions were particle size,120mesh; extraction time of 60min, extraction temperature of 50 C, solvent-to-sample ratio,40:1. Under the optimized conditions, and the extraction yield was 16.19%.2. The determining conditions of Folin-Ciocalteu method were optimized: the volume of Folin-Ciocalteu, 1.00mL; 7.5g/100mL Na2CO3 solution,3.00mL; the reaction temperature,25℃; the reaction time,120 min; and the determination wavelength,647nm. There is a good relationship between the content of gallic acid and the absorbance value within the range of 10-100μg/mL(y=0.0081x-0.0249, R2=0.9996). The suitable concentrations for determining of the methanol, ethanol, and acetone extracts are all 1.25g/100mL. The contents of phenol were 64.4,63.6, and 65.7μg gallic acid/mL, respectively.The optimum conditions for determining the scavenging DPPH·free radical were:515nm,30min. At the concentration of 2.00% for methanol, ethanol, and acetone extracts, the highest free radical scavenging activity were 91.20%,91.79%%,89.59%, respectively.3. The results for purifying of extracts by resins of D301 and XDA-1 were as follows:The results for Static equilibrium adsorption isotherms showed: the adsorptions by D301 resin for methanol, and ethanol extracts are all endothermic process, while an exothermic adsorption process for acetone extracts by XDA-1 resin. The properties of adsorptions by D301 and XDA-1 are all favorable and physical adsorptions. The Freundlich adsorption law is applicable to the adsorptions of extracts on D301 and XDA-1 adsorbents within the concentration in our study.The results for thermodynamics of the adsorptions showed:the adsorption enthalpy change and entropy change of methanol, ethanol extracts on D301 resin are positive, and the free energy change is negative. While the adsorption enthalpy change and free energy change of acetone extracts on XDA-1 resin are negative, and the entropy change is positive.The results for dynamic adsorptions showed 70% ethanol was the suitable de-adsorption solvent, it was good for attaining the adsorption equilibrium at the concentration of 13.5mg/mL, and flow of 2.0mL/min.With the resins of D301, and XDA-1, the purity degrees of methanol, ethanol, and acetone extracts were increased from 10.2%,12.5%, and 14.2% to 34.3%,39.6%, and 37.4%, respectively.4. Experimental values for antioxidant capacity suggested that the extracts possess stronger reduced capacities, inhibition rate on lipid peroxidation reaction induced by Fe2+, and scavenging rate for O2-·and·OH radicals. There is a good relationship between concentrations and effects in the range of experimental concentrations.5. The conditions of HPLC for extracts were:mobile phase,0.1% formic acid-methanol-water; elution gradient,10%→40%→70%→100%→10%(0→10→45→50→60min). The extracts were studied by UPLC/Q-Tof & Mass Spectrometry, and 17 compounds were characterized according to their mass spectrometry, UV/VIS spectrometry, and related literature reports.6. The on-line HPLC screening system was constructed and applied to search for antioxidants from phenolics in Semen Astragali Complanati. The optimized concentration and flow of DPPH were 50mg/L, and 0.70mL/min.There are eight compounds with scavenging DPPH radical activity, whether in methanol extracts, ethanol extracts or acetone extracts. which are 3-O-[2-O-(β-D-glucosyl)-α-L-rhamnosyl] quercetin, 4'-O-(3"'-O-dihydrophaseoyl-β-D-glucopyranosyl) rhamnocitrin, myricetin-3-O-β-D-glucoside, myricetin-5'-O-β-D-glucoside, trimer of flavan with two hydroxyl groups, neocomplanoside, dimer of myricomplanoside, and soyasaponinⅠmethyl ester, and are corresponding to the peaks of chromatogram from number 1 to 8, respectively.7. The off-line HPLC screening results suggested that there are 11 compounds with scavenging DPPH radical activity, either in methanol or ethanol extracts. The compounds for peaks from number 1 to 8 in chromatogram are corresponding to that of 8,1-7 in chromatogram of on-line HPLC screening. One was identified for 3-O-[6-O-(α-L-rhamnosyl)-β-D-glucosyl] isorhamnocitrin in peaks of number 9,10, and 11, and the others were unidentified.There are 12 active peaks in chromatogram of acetone extracts, to which 10 compounds were identified. Peak 1and peak 2 are corresponding to trimer of calycosin, and trimer of kaempferol, respectively. The compounds for the 8 peaks from number 3 to 10 are corresponding to soyasaponin I methyl ester,3-O-[2-O-(β-D-glucosyl)-a-L-rhamnosyl] quercetin,4'-O-(3'"-O-dihydrophaseoyl-β-D-glucopyranosyl) rhamnocitrin, myricetin-3-O-β-D-glucoside, myricetin-5'-O-β-D-glucoside, trimer of flavan with two hydroxyl groups, neocomplanoside, and dimer of myricomplanoside, respectively.