Curcumin Extraction And Interaction With Bovine Serum Albumin

Curcuma longa is a kind of conventional Chinese medicinal material,which is usually used for activating collaterals to alleviate pains,expelling wind-damp,purifying the blood and so on.A large number of researches confirm that curcumin is the major active components of Curcuma longa.Curcumin possess a lot of biological activities and pharmacological effects,such as anti-inflammatory effect,anti-oxidant,anti-cancer,hepatorenal protection and treating Alzheimer’s disease.So this paper will use the Curcuma longa as raw material to research the technology of preparing high purity curcumin using the ultrasonic-assisted aqueous two-phase extraction(UA-ATPE)and semi-preparative HPLC.And this paper also studies the interaction mechanism of bovine serum albumin(BSA)with curcumin,which will provide some theoretical reference for explicating the pharmacokinetics and pharmacological action of curcumin.The main research contents and results as follows:1.This study investigated a new and easy-to-industrialized extracting method for curcumin using ultrasonic extraction technology combined with ammonium sulfate/ethanol aqueous two-phase system(ATPS),and the preparation of curcumin using the semi-preparative HPLC.Firstly,the distribution experiment proved that the optimal ATPS composition was 28%ethanol and 19%(NH4)2SO4.And the single-factor experiments was utilized to determine the material-solvent ratio(g/g),ultrasonic intensity(W/cm2),ultrasonic time(min)and ultrasonic temperature(℃).The material-solvent rate of 3:100,ultrasonic intensity of 30W/cm2,ultrasonic time of 15min and ultrasonic temperature of 25℃ were elected,among them the effect of temperature was not significant.Then the optimum extraction conditions were selected after the response surface methodology(RSM)experiment,which involved material-solvent rate of 3.29:100,ultrasonic intensity of 33.63W/cm2 and ultrasonic time of 17min.At these conditions,the value of extraction yield was 46.91mg/g.2.The extraction yields and purity of curcumin remained stable in the case of amplification,which indicated that scale-up extraction of UA-ATPE was feasible and efficient.Compared with the conventional extracted technology,the UA-ATPE was a time-saving,less solvent and non-toxic extraction method,which eliminated petroleum-based solvents,reduced post-treatment of waste water and ensured a safe product.3.After the semi-preparative HPLC experiment,the optimal preparing condition was determined that mobile phase was acetonitrile:10.5%(V/V)aqueous acetic acid =45:55,injection volume was 50μL,flow rate was 3.5mL/min,stationary phase was C18 column(10μm,10×250mm),detection wavelength was 425nm,column temperature was 35℃.Using these optimal preparing conditions,the curcumin could be completely separated with the demethoxycurcumin and bisdemethoxycurcumin,and the purity of the prepared curcumin could up to 85.58%.4.Fluorescence quenching was used to study the potential interaction mechanism of bovine serum albumin(BSA)with curcumin.The experimental results indicated the mechanism between them was static quenching,not dynamic quenching.And the binding parameters for the interaction of BSA with curcumin were calculated by using the fluorescence quenching measurement.The binding constant(KA)values of them were 17.54×105L/mol(at 298K),9.37×105L/mol(at 306K)and 5.02×105L/mol(at 313K),respectively.The thermodynamic analysis implied that the intermolecular forces between BSA and curcumin were Van der Waals interaction or hydrogen bond,because the values of △H and △S between them were negative.And the interaction process was enthalpy-driven and exothermic,since their AG values were all negative.Synchronous fluorescence spectroscopy suggested that the conformation of tryptophan residue of BSA was changed in the presence of curcumin,because the position of the maximum emission wavelength had a discernable red shift when △λ=60nm.