Screening Co-crystals Based On The Oleanolic Acid And Study On Its Interaction With Bovine Serum Albumin By Fluorescence Spectrometry

Oleanolic acid is a five ring triterpenoid,which has active functions of anti-inflammatory,antioxidant,anti-tumor,protect liver.However,because of its strong hydrophobicity,the gastrointestinal absorption and utilization rate is low,which affects the clinical efficacy.The drug co-crystal is a new solid form of which the active component(API)and the co-crystal former(CCF)are assembled with hydrogen bonds,which is a new technology and a new method to improve the physicochemical properties of API.Objective: In this study,we used oleanolic acid as the API,and the biocompatible small molecules as the CCF to screen the co-crystal of oleanolic acid.Moreover,the interactions between the oleanolic acid co-crystal and bovine serum albumin(BSA)were investigated from molecular level in the simulated physiological conditions,which can provide a theoretical basis for the transport and mechanism of drug co-crystal in vivo.Methods:1 Used Oleanolic acid as API,and benzoic acid,malonic acid,urea pyrimidine,amino acetic acid,para amino benzoic acid,citric acid,succinic acid,saccharin and L-proline as the CCF,co-crystal were screened by ethanol assisted grinding method.The as-prepared co-crystal were characterized by infrared spectroscopy(IR),scanning calorimetry(DSC)and X-ray powder diffraction(XRD).2 Base on the co-crystals screened above,UV-visible spectrophotometry and fluorescence spectroscopy were used to investigate the interaction between oleanolic acid-CCF co-crystals and BSA under the physiological conditions at different temperature.Stern-Volmer equation and modifiedStern-Volmer equation were used to calculate the quenching parameters,including quenching constant,binding constant,binding sites.Furthermore,the interaction mechanism of oleanolic acid co-crystal to BSA was also discussed.Results:1 The XRD patterns and DSC curve of the complexes of oleanolic acid and succinic acid,saccharin,L-Proline,repectively,were significantly different with intact oleanolic acid and corresponding CCF.New peaks appeared in the XRD pattern and DSC curve indicated the formation of co-crystal.2 The interaction of oleanolic acid and three kinds of oleanolic acid co-crystals with BSA were investigated by fluorescence spectrum.At 298 K,the quenching constants of oleanolic acid,oleanolic acid-saccharin co-crystal,oleanolic acid-succinic acid co-crystal,oleanolic acid-L-proline co-crystal were 5.405×103 L·mol-1,2.4×104 L·mol-1,2.01×104 L·mol-1,1.48×104 L·mol-1,respectively;and at 304 K,the quenching constants were 8.958×103 L·mol-1,2.61×104 L·mol-1,1.10×104 L·mol-1,1.56×104 L·mol-1,respectively;and at 310 K,the quenching constants were 1.17×104 L·mol-1,3.80×104 L·mol-1,2.24× 104 L·mol-1,2.23 × 104 L·mol-1,respectively.At 298 K,the binding constant of oleanolic acid,oleanolic acid-saccharin co-crystal,oleanolic acid-succinic acid co-crystal,oleanolic acid-L-proline co-crystal were 3.69× 104 L·mol-1,2.33 × 102 L·mol-1,2.04 × 104 L·mol-1,2.67 L·mol-1,respectively.The thermodynamic parameters of Gibbs free energy(ΔGo),enthalpy(ΔHo),entropy(ΔSo)of interaction between oleanolic acid and BSA were-17.23 k J·mol-1(298K),51.05 k J·mol-1,260.95 J·mol-1·K-1,respectively.Among three kinds of co-crystal system,the binding constant for oleanolic acid-L-proline co-crystal increased with temperature;and the the thermodynamic parameters of Gibbs free energy(ΔGo),enthalpy(ΔHo),entropy(ΔSo)were-3.78 k J·mol-1(298K),265.28 k J·mol-1,902.90 J·mol-1·K-1,respectively.Conclusions:1 Succinic acid,saccharin,L-proline can be used as CCF to prepare oleanolic acid-CCF supramolecular co-crystal system.2 After formation the co-crystal,the quenching constants,binding constants and thermodynamic parameters of interaction between API and BSA changed.The change of the quenching constant and binding constant for oleanolic acid-saccharin and oleanolic acid-succinic acid co-crystals were no regularity when teperature changed,but for oleanolic acid-L-proline co-crystal,the binding constant with BSA increased with the temperature.The fluorescence quenching of oleanolic acid and the co-crystals to BSA was a dynamic quenching.The ΔHo and ΔSo of interaction between oleanolic acid-L-proline co-crystal and BSA were more than zero,so the main force was hydrophobic interaction force.In co-crystal system,the introduction of CCF and the kinds of CCF affected the interaction between API with BSA.