| In this paper,red raspberry(Rubus ideaus L.)fruits were used as the research object,and the anthocyanins in red raspberry fruits were separated and purified by high-speed countercurrent chromatography.The extraction conditions and high-performance liquid chromatography conditions were optimized,and the effective method of separation and purification of anthocyanins in red raspberry fruits were established.Secondly,the molecular interaction between human serum albumin(HSA)and cytarabine was studied by UV–vis absorption,circular dichroism(CD),fluorescence spectroscopy and molecular docking method.1.Isolation and purification of anthocyanins from red raspberry(Rubus ideaus L.)fruits by high-speed counter-current chromatographyAnthocyanins are a sort of important naturally occurring pigments in plants which have many pharmacological effects.Red raspberry fruits have a high content of anthocyanins,which can be used as the source of pigments and have high economic value.However,since anthocyanins are ionic compounds,they are extremely unstable and the polarity of them is relatively large,so there are many difficulties in separating anthocyanins using conventional separation methods.As a liquid-liquid chromatographic separation method,high-speed counter-current chromatography(HSCCC)has no irreversible adsorption and can reduce the loss of sample.It is a commonly used separation method of separating anthocyanins.The purpose of this research was to explore a method for efficient and rapid isolation and purification of anthocyanins from red raspberry fruits by HSCCC.The results obtained are as follows:In this research,a method of ultrasonic extraction as extracting anthocyanin method,macroporous resin as purifing anthocyanin method combined with HSCCC for isolation and purification of three anthocyanins in red raspberry fruits was established.In the ultrasonic extraction method,the ratio of material to liquid was 1 g:10 mL,and the extraction solution was 80%(v/v),pH 2.0 ethanol aqueous solution,and the ultrasonic extraction temperature was 35°C,and the ultrasonic extraction time was 50 min;In the macroporous resin purification method,the macroporous resin was AB-8,and the eluent solution was 80%(v/v)ethanol aqueous solution;In the HSCCC separation method,a two-phase solvent system consisting of n-butanol,methyl tert-butyl ether,acetonitrile,water and trifluoroacetic acid(5:1:1:4:0.001,v/v)was obtimized to separation anthocyanins from red raspberry fuits.the injection volume was 150 mg samples/15 mL solvent system,and the flow rate of the mobile phase was 2.0 mL/min,the rotation speed was 850 r/min.Finally,three anthocyanins were successfully isolated from red raspberry fruits:cyanidin-3-O-sophoroside,cyanidin-3-O-(2~G-O-glucosylrutinoside)and cyanidin-3-O-glucoside,and their purity was 95.3%,91.3%and 99.1%,respectively.Among them,cyanidin-3-O-glucoside and cyanidin-3-O-glucoside were firstly isolated from red raspberry by HSCCC.And cyanidin-3-O-(2~G-O-glucosylrutinoside)was firstly isolated from red raspberry.This study developed an effective method for separating high purity anthocyanins from red raspberry fruits,which has the advantages of high efficiency,rapidity and simplicity.This study could provide new ideas and theoretical basis for separating ionic and polar compounds such as anthocyanins from other plants and would lay the foundation for industrial production of anthocyanins in red raspberry fruits and further promote the development and application of anthocyanins in food,cosmetics,medicine and health care products,and increase the economic application value of anthocyanins.2.Study on the molecular interaction between HSA and cytarabineIn this research group,a large number of anti-cancer drugs were investigated.Two small molecular compounds with anticancer activity,cytarabine and catechin,were selected as the research objects of this subject,using spectroscopy and molecular docking techniques.Simultaneously,in order to simulate the interaction between the drug and human serum albumin better after absorption of drugs into the blood,the interactions between catechin and human serum albumin in glucose free environment and normal human blood glucose concentration environment were compared to explore the effect of normal human blood glucose concentration environment on the interaction between small molecular compounds and human serum albumin.The main research results are as follows:The results showed that the small molecular compounds cytarabine and catechin interacted with HSA to cause fluorescence quenching of HSA,and the fluorescence quenching mechanisms of cytarabine and catechin were static quenching.Cytarabine interacted with HSA via site I of HSA and catechin interacted with HSA via site II.The thermodynamic studies showed that the cytarabine interacted with HSA mainly through van der Waals force and hydrogen bonds;while in glucose free environment,catechin interacted with HSA mainly through hydrophobic interaction,and in glucose environment catechin interacted with HSA mainly through van der Waals force and hydrogen bonds.The binding distance of cytarabine calculated was 3.4 nm and the F?rster no-radiation energy transfer of cytarabine was occurred.In glucose free environment,the binding distance of catechin calculated was 2.6 nm and the F?rster no-radiation energy transfer of catechin was occurred.In addition,In glucose environment,the binding distance of catechin calculated was 2.5 nm and the F?rster no-radiation energy transfer of catechin was occurred.In addition,the results of the three-dimensional(3D)fluorescence spectra indicated that the addition of cytarabine and catechin changed the conformation of HSA.The results of circular dichroism(CD)showed that cytarabine and catechin could change the conformation of secondary structure in HSA.The results of molecular docking further confirmed that cytarabine binds to the HSA via site I by van der Waals force and hydrogen bonds.It suggested lysine residue K199,histidine residue H242,arginine residue R257 and alanine residue A291 of HSA combined cytarabine with hydrogen bonds and arginine residue R222,leucine residues L238 and L260,serine residue S287,alanine residue A261 and tryptophan residue W150 bound cytarabine via van der Waals force,which has not been reported before.The results of molecular docking further confirmed that catechin binds to the HSA via site II in glucose free environment by hydrophobic interaction,it suggested alanine residues A215,A217,proline residues V216,Val235,phenylalanine residues F223,F221 of HSA combined catechin via hydrophobic interaction,which has not been reported before;and catechin binds to the HSA in glucose environment by van der Waals force and hydrogen bonds,it suggested lysine residue K199,tryptophan residue W214,arginine residue R218,serine residue S220,leucine residue L219,arginine residue R222,lysine residue K286 of HSA combined catechin with via van der Waals force and hydrogen bonds,which has not been reported before.In this experiment,we also investigated the interaction between catechin and HSA in simulated normal human blood glucose environment and glucose free environment.The results showed that the addition of catechin in the glucose environment and glucose free environment could introduce the fluorescence quenching of HSA and the quenching mechanisms are static quenching.while the glucose environment might influence the interaction of HSA with catechin via changing the conformation of HSA,such as binding force and other aspects.The interactions between catechin and human serum albumin in glucose free environment and normal human blood glucose concentration environment were compared to explore the effect of normal human blood glucose concentration environment on the interaction between small molecular compounds and human serum albumin,and this research would have certain guiding significance for next research of the interaction between small molecular compounds and human serum albumin.The research are helpful to understand of bioavailability and the processes of distribution,metabolism,excretion,which is conducive to better clinical guidance medication,and improve the safety of using drugs.It also provides some reference for its pharmacokinetics and its pharmacological and pharmacodynamic theoretical significance. |
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