Study On The Isolation And Purification Of Polyphenolics From Sorbus Pohuashanensis Hedl.fruits By High-speed Counter-current Chromatography And The Interaction Of Several Small Molecular Compounds With Human Serum Albumin

1.Isolation and purification of polyphenols from Sorbus pohuashanensis Hedl.fruits by High-speed Counter-current ChromatographySorbus pohuashanensis Hedl.(S.pohuashanensis)is a member of family Roseceae.The fruits are eatable and have been commonly used as a medicine.Pharmacological research has indicated that S.pohuashanensis fruits exhibit a wide range of biological activities,such as anti-tussive,anti-asthmatic,anti-inflammatory,anti-oxidation,anti-radiation and anti-cancer.Moreover,S.pohuashanensis fruits are rich in flavanols and phenolic acids which are main active constituents of it.High-speed counter-current chromatography(HSCCC)is a new-type liquid-liquid partition chromatographic technique.It does not need the solid carrier and can completely eliminate the problems of contamination,denaturation,irreversible adsorption of the sample.In addition,HSCCC provides diverse advantages such as low-cost,simple operation and high recovery and so on.In this study,HSCCC was used to separate and purify polyphenolic compounds from S.pohuashanensis fruits.In this work,ultrasonic extraction is used to obtain crude extracts.The gradient elution was optimized on a polyamide chromatography column to obtain 50%and70%eluted fractions.And then,the optimized liquid chromatography conditions were applied to analyze the components in S.pohuashanensis fruits and select the two-phase solvent systems.The partition coefficients(K),the separation factor(?)and retention of stationary phase(S_F)are key factors of the selection of two-phase solvent system.Based on these parameters,the optimum system were ethyl acetate-n-butanol-water3.5:1.5:5(50%fraction)andn-hexane-ethyl acetate-methanol-water 1:3:1:3.5(70%fraction).Then,the parameters of HSCCC were investigated.The results showed that 50%fraction and 70%fraction had the best separation effect when the injection volume was 150 mg,the rotation speed was850 r/min,and the flow rate was 2.0 mL/min.Five polyphenols,including neochlorogenic acid(30.2 mg),chlorogenic acid(28.5 mg),quercetin 3-O-(6?-?-L-rhamnopyranosyl-4'''-?-L-rhamnopyranosyl)-?-D-glucopyranoside(7.5mg),3,5-O-dicaffeoylquinic acid(6.3 mg),and rutin(9.4 mg),were successfully isolated with high purities(over 95%)by only one-step of HSCCC separation.Among the five components,the other four compounds except rutin were isolated from S.pohuashanensis fruits for the first time.Among them,chlorogenic acid and neochlorogenic acid were isomers,and quercetin 3-O-(6?-?-L-rhamnopyranosyl-4'''-?-L-rhamnopyranosyl)-?-D-glucopyranoside and 3,5-O-dicaffeoylquinic acid were first identified and isolated from Sorbus fruits.In the study,we successfully established an effective and fast method to separate and purify polyphenolic compounds from S.pohuashanensis fruits.The research will lay the foundation for in-depth research and industrial production of active constituents in S.pohuashanensis fruits,and promoted its development and application in food,medicine,cosmetics and other fields.2.Study on the interaction between several small molecular compounds and human serum albuminTussilagone is an active sesquiterpenoid of The flower bud of Tussilago farfara L.which exhibits a variety of pharmacological activities,such as anti-tussive,anti-asthmatic,lipid-lowering,anti-inflammatory and anti-allergic,etc.At present,our research group has conducted the study on the pharmacokinetics and in vitro and in vivo metabolism of the tussilagone.In order to make the research of the in vivo process of the tussilagone more comprehensive and scientific,we firstly carried out the study on the interaction of tussilagone with human serum albumin(HSA).Hyperoside,which has similar pharmacological activities,is an important flavonol glycoside compound with antitussive,anti-inflammatory,anti-oxidation,anti-allergic,analgesic,liver-protecting and anti-tumor effects.In this paper,we also studied the interaction between hyperoside and HSA,and discussed the effect of vitamin C(VC)on the interaction between hyperoside and HSA.Through the investigations of the quenching type,number of binding sites(n),binding distance(r),binding constant(Ka),binding sites of tussilagone-HSA and hyperoside-HSA and conformational changes of HSA,the researches are helpful to understand of bioavailability and the processes of distribution,metabolism,excretion.Meanwhile,the study will provide guidance for the pharmacokinetics and clinical use of tussilagone and hyperoside.It provides a reference for exploring the drug-protein interaction rules in complex systems.In this study,three-dimensional spectra,circular dichroism(CD)spectra,fluorescence spectroscopy,UV-vis absorption,molecular docking techniques were used to investigate the interaction of tussilagone-HSA,hyperoside-HSA,hyperoside-VC-HSA by double logarithmic equation,Stern-Volmer equation,F?rster non-radiative energy transfer theory and Van't Hoff equation.The results indicated that the fluorescence quenching of HSA-tussilagone and HSA-hyperoside were a static quenching process as the result of HSA-tussilagone(1:1)and HSA-hyperosde(1:1)complex.Tussilagone spontaneously bound to HSA in site I based on hydrophobic forces and hydrogen bonds,and hyperoside spontaneously bound to HSA in site I based on hydrophobic forces.In the presence of VC,the interaction type between hyperoside and HSA was changed to van der Waals force or hydrogen bond.In addition,VC also reduced the binding constant of hyperoside to HSA,that was,when VC and hyperoside coexisted,VC decreased the stability of the combination of hyperoside and HSA.The binding distance of tussilagone and hyperoside were estimated to be 2.07 nm and 2.49 nm respectively,which indicated the occurrence of fluorescence energy transfer in HSA-tussilagone and HSA-hyperosde.However,when VC existed,the binding distance of HSA-hyperosde was increased to 2.75 nm.The results of three-dimensional fluorescence spectra and circular dichroism spectra shown that the conformation of HSA induced by tussilagone and hyperoside was changed,and the presence of VC will further alter the conformation of HSA in the hyperoside-HSA system.Moreover,the molecular docking technique was used to obtaintheaminoacidresiduesofaspartame-HSA,hyperoside-HSA,hyperoside-VC-HSA at the molecular level and the sites of interaction with HSA,which provided evidence for the above studies.