Study On The Purification And Bioactivities Of Flavonoids In Sophora Flavescens Ait.

The dried root of Sophora flavescens Ait.（Leguminosae）is a traditional Chinese medicine well known for its efficacy of eliminating heat,dehumidification,desinsectization and diuresis.S.flavescens Ait.show unique pharmacological effects such as antioxidation,bacteriostasis and anti-inflammation by treating many diseases with other drugs.Notably,its antioxidant activity has become one of the focuses of current research.Alkaloids and flavonoids are the main constituents of S.flavescens Ait..To date,matrine and oxymatrine are the qualitative and quantitative indexes of S.flavescens Ait.in pharmacopoeia of people’s republic of China.However,matrine and oxymatrine are also the qualitative and quantitative indexes of S.tonkinensis Gagnep.,which has different clinical efficacy compared with S.flavescens Ait..It suggested that it′s imperfect to evaluate the quality and efficacy of S.flavescens Ait.only alkaloids are studied.The pharmacodynamic material bases of S.flavescens Ait.need further study.Our previous research found that the HPLC fingerprint of flavonoids in S.flavescens Ait.could effectively distinguish S.flavescens Ait.and S.tonkinensis Gagnep.,which suggested that flavonoids of S.flavescens Ait.had a certain specificity.However,there is little study on the isolation and purification of specific flavonoids from S.flavescens Ait..In addition,the quality of standard substances of S.flavescens Ait.flavonoids sold in markets are irregularity with a high price.Thus,how to establish a more efficient isolation method combining with the antioxidant property of flavonoids is the key point of the further study on flavonoids from S.flavescens Ait..Simaltaneously,evaluations of the antibacterial and anti-inflammation activities of flavonoids from S.flavescens Ait.are conducive to improve its contribution to the traditional efficacy of S.flavescens Ait..In this paper,TLC-DPPH,column chromatographies and recrystallizations were used to isolate the main flavonoids of S.flavescens Ait..Simultaneously,bioassay guided isolation and rapid detection methods of flavonoids from S.flavescens Ait.were established by NMR,UPLC-Q-TOF-MS and HPLC-DAD.In addition,a preliminary study on the bioactive of（total）flavonoids from S.flavescens Ait.were conducted via in vitro antibacterial,in vivo and in vitro anti-inflammatory experiments.Part 1,study on the chemical constituents of flavonoids from S.flavescens Ait.based on a TLC-DPPH guided isolation.First,TLC-DPPH method was used to screen the antioxidant activity components of ethyl acetate extracts from S.flavescens Ait.,and it was used to compare the antioxidant capability of 15 S.flavescens Ait..Then,it was not until S.flavescens Ait.extracted with 95%ethanol that flavonoid fractions were obtained by combining with kieselguhr-ethyl acetate method.Subsequently,12 antioxidant activity ingredients were isolated from S.flavescens Ait.via bioactivity-guided isolation combining with different column chromatographies and recrystallizations.They were identified as trifolirhizin,nor-anhydroicaritin,calycosin,isoxanthohumol,trifohrhizin-6′-monoacetate,kurarinone,sophoraflavanone G,maackiain,formononetin,8-lavandulyl-5,7,4′-trihydroxy-flavonol and 5-methyl-kushenol C via MS and NMR data analysis.KS-12 is an unknown flavonoid needing further study.It should be noticed that based on a TLC-DPPH guided isolation,more than 2 g of kurarinone and sophoraflavanone G were got.Simultaneously,LODs measured by direct TLC-DPPH was conducted to compare the antioxidant activity of11 flavonoid monomers mentioned above,and results showed that 5-methyl-kushenol C（<<0.06 mM）>kurarinone,sophoraflavanone G（<0.06 mM）>isoxanthohumol,calycosin（0.06 mM）>8-lavandulyl-5,7,4′-trihydroxy-flavonol,trifohrhizin-6′-monoacetate,maackiain（0.12 mM）>nor-anhydroicaritin,formononetin（0.25 mM）>trifolirhizin（0.5mM）.Then,UPLC-Q-TOF-MS was established to qualitatively evaluate the flavonoids of S.flavescens Ait..Simultaneously,HPLC-DAD was utilized to quantitatively determine the contents of kurarinone and sophoraflavanone G from S.flavescens Ait.extracts.Part 2,in vitro antibacterial study of flavonoids from S.flavescens Ait..In this part,MICs,LODs and diameters of inhibition zone were measured by broth microdilution method,direct TLC-bioautography-MTT method and disk diffusion method,respectively,to study the antimicrobial activity of SA,SF and their monomers（matrine,oxymatrine;kurarinone,sophoraflavanone G,5-methyl-kushenol C,maackiain and trifolirhizin）against different bacterias.The results showed that the MICs of SF against Escherichia coli and Staphylococcus aureus were 1 mg/mL and 0.25 mg/mL,respectively;SA′s were 32 mg/mL and 8 mg/mL,respectively.The LODs of alkaloid monomers（matrine,oxymatrine）against Escherichia coli and Staphylococcus aureus were both>4 mM,while flavonoid monomers′（kurarinone,sophoraflavanone G,5-methyl-kushenol C）were<0.3 mM and 0.5¹ mM,respectively.Results of inhibition zone showed that sophoraflavanone G had the strongest antimicrobial activity against the testing bacteria,followed by kurarinone and maackiain,while trifolirhizin engendered no effect.All of them had no effect on Pseudomonas aeruginosa.On balance,the antimicrobial activity of SF was stronger than SA.Part 3,in vivo and in vitro anti-inflammatory study of flavonoids from S.flavescens Ait..In this part,LPS-induced RAW264.7 cells and three pharmacological models including dimethybenzene-induced auricle edema,acetic acid-induced peritoneal permeability and carrageenan-induced hind paw edema in mice were utilized to study the anti-inflammation effects of SA and SF from S.flavescens Ait..Results of dimethylbenzene-induced auricle edema showed that SF presented obvious anti-inflammation effect at the dose of 100 mg/kg and 200 mg/kg,while SA presented at the dose of 200 mg/kg.It confirmed that the high dose group of SF had the strongest anti-inflammation effect and its inhibition ratio was 41.2%.Results of acetic acid-induced peritoneal permeabilityc showed that SF presented significant difference at the dose of 100 mg/kg,SA and SF presented very significant difference at the dose of 200 mg/kg.The inhibition ratios of SA and SF at the dose of 200 mg/kg were 39.9%and 50.2%,respectively.Results of carrageenan-induced hind paw edema showed that SA and SF presented significant difference after infected 4 h and 6 h at the dose of 100 mg/kg,and the inhibition ratios of SA and SF were 52.8%and59.4%,respectively,after mice were treated 6 h at the dose of 100 mg/kg.SA and SF presented very significant difference after infected 1 h,2 h,4 h and 6 h at the dose of 200mg/kg,and the inhibition ratios of SA and SF were 74.4%and 72.0%,respectively,after mice were treated 6 h at the dose of 200 mg/kg.Results of PGE₂ determination indicated that compared with indomethacin groups,SA and SF presented very significant difference at the dose of 200 mg/kg.In vitro experimental results showed that SF and SA did not have cytotoxic effect on RAW264.7 cells within the range of 100μg/mL.Compared with LPS and SA groups,SF group（25,50,100μg/mL）could significantly and dose-dependently inhibit the release of pro-inflammatory cytokines NO,TNF-α,IL-6 and MCP-1 on LPS-induced RAW264.7 cells,which indicated that SF had a stronger anti-inflammatory effect than SA.In summary,TLC-DPPH,column chromatographies and recrystallizations were used to isolate flavonoid monomers of S.flavescens Ait..Simultaneously,bioassay guided isolation and rapid detection methods of flavonoids from S.flavescens Ait.were established by NMR,UPLC-Q-TOF-MS and HPLC-DAD.In addition,in vitro antibacterial,in vivo and in vitro anti-inflammatory experiments were conducted to study whether the flavonoids make a contribution to the traditional efficacy of S.flavescens Ait..What we have done could provide scientific basis for the further study of S.flavescens Ait..The innovation of this paper is as follows:1.TLC-DPPH guided isolation,a new isolation method,was established to isolated the flavonoids of S.flavescens Ait..2.A preliminary study on the bioactive of flavonoids from S.flavescens Ait.were conducted via in vitro antibacterial,in vivo and in vitro anti-inflammatory experiments,compared with the activities of alkaloids.And it preliminarily proved that flavonoids of S.flavescens Ait.made a contribution to the traditional efficacy of S.flavescens Ait..