The Separation,Antioxidant Activity And LC-MS Analysis Of Flavonoids And Proanthocyanidins Of Extract From Limonium Sinense(Girard)Kuntz Root

Limonium sinense whose root can be used as medicine is a traditional Chinese drug. The polyphenol extract from the root of L. sinense had strong scavenging capacity on free radical, but its special chemical structure and effect principle of scavenging free radical has not been reported. In this thesis, L. sinense root extract was used as experimental materials, and adopted sequentially polyamide and Sephadex LH-20as the column chromatography medium. Through evaluating systematically the antioxidant activity by three anti-oxidative systems, we got flavonoid and proanthocyanidin fractions with higher antioxidant activity. And then, we analyzed preliminarily their chemical structures by LC-MS. The main results are as follows:1. Ultrasonic-assisted extraction technology and macroporous adsorption resin technology were used to separate and purify polyphenol compounds from roots of L. sinense. Then, it was purified by column chromatography with polyamide, eluting sequentially with50%ethanol,70%ethanol, methanol mixed with ammonia(pH8.0),40%acetone and60%acetone,and finally we obtained nine fractions(frac l-frac9).Analyzing comparatively the antioxidant activities of these nine fractions by DPPH, TEAC and FRAP methods, we found that frac9and frac8conformed to chracteristic spectrum of proanthocyanidin, and their total phenolic contents and antioxidant activities were strong. So they were focused as repurity. In addition, frac2complied with chracteristic spectrum of flavonol, and had a strong antioxidant capacity. So it was focused as repurity, too.2. Proanthocyanidin fractions-frac8and frac9were purified by column chromatography with Sephadex LH-20, eluting sequentially with95%ethanol, methanol and40%acetone, and finally we obtained four fractions (LSPA1, LSPA2, LSPA and LSPA4) from frac8and three fractions (LSPA5, LSPA6and LSPA7) from frac9, respectively. Analyzing comparatively the antioxidant activities of these nine fractions, we got two fractions (LSPA2and LSPA6) which had strong capacities of scavenging radical and ferric reducing/antioxidant power were focused as the main objects of the LC-MS analysis.3. After acid hydrolysis of LSPA2and LSPA6, the solutions became dark red, and had the max absorption peak at540nm. So LSPA2and LSPA6can be identified containing proanthocyanidins. These two acid hydrolyzated fractions were analyzed by LC-MS. Our results demonstrated that they both had a single peak at the same time, MS detection of the substance was (M)303, which is consistent with prodelphinidin. Another, the antioxidant capacity relating to degree of polymerization of proanthocyanidins,that the higher the degree of polymerization, the weaker antioxidant capacity, combined with the strong capacity of scavenging radical and ferric reducing/antioxidant power of LSPA2and LSPA6, we can preliminarily conclude that LSPA2and LSPA6are oligomeric proanthocyanidins.4. The flavonol fraction-frac2was purified by column chromatography with Sephadex LH-20, eluting sequentially with10%ethanol and95%ethanol, and finally we obtained five fractions (LSFL1, LSFL2, LSFL3, LSFL4and LSFL5) from frac2. Analyzing comparatively the antioxidant activities, we found that LSFL4had a strong capacity of scavenging radical and ferric reducing/antioxidant power. LSFL4was focused as the main object of the LC-MS analysis.5. There were8peaks of LSFL4at254nm and360nm by HPLC analysis. By MS analysis, we found (M-H)-of peak1and peak2were463.0889and447.0934, suggesting the molecular formula were C21H20O12and C21H20O11, respectively.(M-H)-of peak3and4were505, suggesting the molecular formulas were C23H22O13, may isomers;(M-H)’of peak7and peak8were547, presuming the molecular formulas were C25H24O14. Other peaks were unable to form a high matching formula by software. LSFL4which was acid hydrolyzated detected two aglycones by LC-MS, and their (M-H)+were respectively319.0446and303.0503. LSFL4acid hydrolyzate LC-MS detected two aglycone, the (M-H)+. We determined the aglycones of flavonoid fraction-LSFL4were myricetin and quercetin, their molecular formulas were respectively C15H10O8and C15H10O7.6. According to the estimate of software, peak1, peak2, peak3, peak7and peak8of LSFL4in a mass spectrometry were determined by two stage mass spectrometry. By MS2analysis, Substance1, Substance3, Substance7and Substance8can be determined myricetin-3-O-glucoside. Substance1may be myricetin-3-O-rhamnopyranoside which was the chemical composition of L. sinense reported. Substance3was presumed myricetin-3-O-β-D-glucose-6"-acetate, Substance7may be Myricetin-3-O-(3,6-diacetylgalactoside) and Substance8can not determine the specific molecular structure. Substance2can be determined quercetin-3-O-glucoside, and was presumed quercetin-3-O-a-L-rhamnopyranoside.