Purification Of Flavonoids From Glycyrrhiza Glabra L. Leaf And Their Biological Activities And Applications

Licorice is a kind of herbaceous plant belongs to medicine food homology. It is widely used in prescription of Traditional Chinese Medicine and food industry. Nowadays, the root and rhizomes of licorice are widely used, while the aerial part is used as feed for cattle and flock, or burnt into fertilizer as the fuel with less value. There are quite abundant researches on the constituent and biological activity of licorice root and rhizomes, but little information about the aerial part. The chemical profile and biological activity of Glycyrrhiza Glabra L. root and leaf were compared, to ensure the research value on the leaf. Flavonoids in G. Glabra L. leaf were purified and identified in terms of chromatographic and spectroscopic techniques, and their biological activities were evaluated. The measure and extraction method of flavonoids in G. Glabra L. leaf were optimized, and macroporous resins were used to enrich the flavonoids. The inhibitory effects of G. Glabra L. leaf flavonoids on oil oxidantion and myofibrillar protein oxidation in pork during the storage time were studied, to provide theory guide for the high-value utilization of G. Glabra L. leaf. The research contents are as follows:（1） HPLC was used to analyze the differences of chemical profile and its content in ethanol extracts of Glycyrrhiza Glabra L. root, stem and leaf. Compared the total flavonoid content, ORAC value, DPPH scavenging capacity, nitrite scavenging capacity and tyrosinase inhibitory effect of G. Glabra L. root and leaf extracts, and confirmed the superiority of leaf. Flavanone pinocembrin and liquiritin were separately confirmed as the major flavonoid in G. Glabra L. leaf and root. Pinocembrin exhibited better antioxidant activity and nitrite scavenging capacity but moderate inhibitory effect on mushroom tyrosinase compared with liquiritin. Results from cell antioxidant activity assay showed that pinocembrin and liquiritin had significant protection effect on H₂O₂^-injured PC12 cells at a low concentration（3.125 μM）. Pinocembrin in high concentration（≥100 μM） promoted the cell growth while liquiritin in high concentration（≥100 μM） caused the cytotoxic effect.（2） Five flavonoids with similar chemical structures were isolated from G. Glabra L. leaf and identified as glabranin, pinocembrin-7-O-β-D-glucopyranoside, pinocembrin, pinobanksin and galangin. Pinobanksin was firstly isolated from Glycyrrhiza plants（even from leguminous plants）. A method, which could obtained high purity pinocembrin only through enriching by polyamide resin, dissolving in ethanol and precipitating in distilled water, and then recrystallizing in ethanol, was established. The inhibitory effects of five flavonoids against xanthine oxidase（XOD） were determined in a decreasing order of: pinobanksin > galangin > pinocembrin > pinocembrin-7-O-β-D-glucopyranoside > glabranin. Fluorescence quenching studies suggested that these flavonoids could interact with XOD at more than one binding sites. The Lineweaver-Burk and Dixon plots illustrated the inhibitory mechanisms underlying varied with type and concentration of substrate and inhibitor. Docking results showed that galangin and pinobanksin could be inserted into the active site of XOD, while still forming hydrogen bonds with amino acid residues such as Ser-876, Asn-768, Glu-1261 and Thr-1010 and sandwiching aromatic interactions（π-Ï€ effects） around the active site of XOD to increase the stability of the flavonoids-XOD combination.（3） A method, which determined the total flavonoids of G. glabra L. leaf rapidly and accurately, was optimized. The standard, coloration method and determination wavelength were determined as quercetin, Al Cl₃-CH₄ O coloration and 314 nm, respectively. Ethanol was chosen as the extraction solvent based on preliminary experiment. Ultrasonic was used to assistantly extract G. glabra L. leaf flavonoids. The flavonoids extraction yield and DPPH scavenging capacity of extract were chosen as index. Response surface method was used to optimize the ultrasonic extraction process.（4） The kinetics of adsorption and desorption behaviors of five macroporous resins（XAD-4, XAD-16, HP-2MGL, SP-825 and SP-207） for enriching flavonoids from G. glabra L. leaf were investigated. All the five resins could adsorb G. glabra L. leaf flavonoids rapidly. Pseudo-second-order kinetics model was suitable for evaluating the whole adsorption process and the adsorption process was influenced by surface diffusion and particle diffusion together. Additionally, Freundlich model could evaluate the isothermal adsorption of two representative resins（XAD-16 and SP-825） better, and the adsorption of the representative resins was an exothermic and physical adsorption process. Further column chromatography of XAD-16 and SP-825 showed that the total flavonoids were enriched in 70% and 90% ethanol fractions. Meanwhile, the antioxidant capacities and nitrite scavenging capacities of different fractions showed obvious correlation. However, the antioxidant capacities and nitrite scavenging capacities of different fractions showed no significant correlation with their flavonoids and pinocembrin contents.（5） The inhibitory effects of pinocembrin and G. glabra L. leaf flavonoids（XAD-16-50% constituent） on oil oxidation and myofibrillar protein oxidation in pork products during the storage time were evaluated. Results showed both pinocembrin and G. glabra L. leaf flavonoids（XAD-16-50% constituent） significantly inhibited the oil oxidation during the cold storage period of ground pork（15 days） and frozen storage period of minced pork cutlets（60 days）. G. glabra L. leaf flavonoids（XAD-16-50% constituent） showed a comparative inhibitory effect under the additive amount of 0.05%（w/w） compared with TBHQ（0.02%; w/w）, and that was better than pinocembrin. Meanwhile, the inhibitory effect of G. glabra L. leaf flavonoids（XAD-16-50% constituent） was not affected by temperature. Both pinocembrin and G. glabra L. leaf flavonoids（XAD-16-50% constituent） showed a certain inhibition on myofibrillar protein oxidation but no significant difference among different additive amount. In this research, the inhibitory effects of the test antioxidants on oil oxidation and myofibrillar protein oxidation showed no significant correlation. From the results of GC-MS, pinocembrin and G. glabra L. leaf flavonoids（XAD-16-50% constituent） could significantly change the content of flavoring substances including aldehydes, alcohols, esters and ketones in raw and cooked minced pork cutlets during the storage time.（6） Oil oxidation stability analyzer was used to evaluate the inhibitory effect of three G. glabra L. leaf flavonoids constituents including XAD-16-30%, XAD-16-50% and XAD-16-70% on oil oxidantion. Results showed they could effectively inhibit the oxidation of lard with obvious dose-effect relationship. Result from temperature extrapolation method indicated G. glabra L. leaf flavonoids（XAD-16-50% constituent） with the additive amount of 0.05%（w/w） could extend the shelf life of lard for 15 days（stored at 20 ℃）.