Study On Fermentation Pretreatment,Extraction And Biological Activity Of Total Flavonoids From Licorice Residues

Licorice is widely distributed in Xinjiang,Inner Mongolia,Tibet and other regions,and is widely used because it is rich in flavonoids and has anti-cancer,antioxidant and anti-inflammatory effects.The extraction of active ingredients in licorice is usually carried out by water extraction method,resulting in wasteful extraction of fat-soluble flavonoids from licorice residues.Licorice residue is rich in lignocellulose,which causes difficult precipitation of active substances.The main methods of traditional pretreatment of lignocellulose are ultrasound-assisted and microwave-assisted,but they are energyconsuming and polluting,so the solid-state microbial fermentation method is chosen for pretreatment of lignocellulose,whose main advantages are low production price,in line with the concept of sustainable development,and industrial production.Therefore,in this paper,solid-state mixed bacterial fermentation method was used to disrupt the structure of lignocellulose and promote the precipitation of total flavonoids in licorice residue,furthermore,natural and green deep eutectic solvents were selected for the extraction of total flavonoids,and the highest content of licorice was obtained by isolation and purification,and its mechanism in anti-cancer cell Hela was investigated,and the following main results were obtained.Using the ability of exo-glucanase,pectinase and laccase as reference,the white rot fungis with high laccase production and the Lichtheimia ramose with high pectinase and exo-glucanase production were selected as the mixed fermentation strains of licorice residues by single fermentation and single-factor experiments.The optimum conditions for solid-state mixed fermentation were obtained: 1:1 inoculation ratio of white rot fungus and Lichtheimia ramose and 10% inoculum,20% urea and ammonium nitrate addition(1:2),and 50% water content.SEM demonstrated that mixed fermentation destroyed the original lignocellulose structure of licorice residues,and FT-IR further demonstrated the removal of wax and reduction of hemicellulose content on the surface of mixed fermentation.The mixed bacterial fermentation reduced the cellulose and hemicellulose contents of licorice residues by 0.7% and 11.3%,respectively,and increased the total flavonoid yield by 75%,and three compounds,glycyrrhizone,isoflavone and glycyrrhiza chalcone A were detected in the extracted total flavonoid compared with unfermented licorice residue.In order to select efficient and green solvents for flavonoid extraction,deep eutectic solvents were screened using COSMO-RS theory,and tetramethylammonium chloride,tetraethylammonium bromide,tetrapropylammonium bromide and tetrabutylammonium bromide as hydrogen bond acceptors and ethylene glycol,lactic acid and propionic acid as hydrogen bond donors were screened as candidate solvents for total flavonoid extraction from licorice residues.Using liquiritigenin as the model compound,tetrapropylammonium bromide as the hydrogen bond acceptor and ethylene glycol and lactic acid as the hydrogen bond donors were optimally identified.The results showed that when tetrapropylammonium bromide and ethylene glycol were used as the extraction solvents with the optimal solvent molar ratio of1:1,water content of 50%,extraction time of 60 min,extraction temperature of 80 ℃ and material-liquid ratio of 1:6,the flavonoid yield was increased by 49.4% compared with the traditional ethanol extraction method,and the scavenging ability of ABTS and DPPH radical scavenging ability was increased by 21.2%and 24.1%,respectively.To study the biological activity of the extracted flavonoids,the total flavonoids were purified by gradient elution through D101 macroporous resin,MCI gel column,ODS column,and Sephadex LH-20,and then the main component liquiritigenin was obtained by LC-MS and NMR identification with a purity of more than 95%.The purified liquiritigenin was applied to study the cytotoxicity,cell cycle,cell morphology and apoptosis mechanism of related gene pathway and protein pathway of cervical cancer cells.The results showed that the cell cycle of cervical cancer cells is G2/M phase,and the effect of liquiritigenin on the toxicity and inhibition rate of cancer cells was significant when the concentration of liquiritigenin was 500-900μg/ml,and the cell morphology and number were wrinkled and changed with the increase of liquiritigenin concentration.The expression of Caspase-8,an important death receptor pathway,was upregulated and induced the upregulation of TNFR1,DR5 and Fas genes.It was shown that the solid state fermentation pretreated licorice residues based on deep eutectic solvent extraction has the best flavonoid yield and antioxidant activity,and after column chromatography separation and purification to obtain pure liquiritigenin has better inhibitory effect and toxicity to cervical cancer cells,and after important apoptotic pathway study showed that the mitochondrial pathway and death receptor pathway interact with each other.