The Research Of Bioavailability Of Oleanolic Acid And β-cyclodextrin Derivatives

Oleanolic acid has a variety of pharmacological activities. However, due to its lowsolubility and poor bioavailability, the application of oleanolic acid is limited. As a classof compounds with a hollow cylindrical structure, cyclodextrins are widely used inpharmaceutics, which are mainly used to improve the solubility of poorly soluble drugs andto improve the bioavailability of the drugs. There are many types of cyclodextrins, and theapplication of β-cyclodextrin is the most prevalent. In recent years, the development of avariety of β-cyclodextrin derivatives provides bright prospects in pharmaceuticalpreparations. To the best of our knowledge, ther is no literature reporting thethermodynamic stability and bioavailability of inclusion compound of oleanolic acid andβ-cyclodextrin derivatives.Objective: The solubility is very low, leading to its poor bioavailability. Theβ-cyclodextrin derivatives could effectively promote the solution of insoluble drugs, and thus improve the absorption and bioavailability. In this study, we aimed to:（1） determinedifferent oleanolic acid/β-cyclodextrin derivatives inclusions using high performanceliquid chromatography;（2） evaluate the thermodynamic stability of the oleanolicacid/β-cyclodextrin derivatives inclusion using phase solubility;（3） examine the vivopharmacokinetic behavior and bioavailability of oleanolic acid/hydroxypropylβ-cyclodextrin inclusion in rats. All of these laied the foundation for oleanolic acidpreparations.Methods:（1） the determination of oleanolic acid/different β-cyclodextrin derivativesinclusion complexs were conducted by a HPLC modified-method. All methodologyvalidation was fulfilled according to the related guideline. The chromatographicconditions were as follows: Agilent ZORBAX SB-C18column （150mm×4.6mm,5μm）was used; the mobile phase was composed of methanol, acetonitrile and water whoseratio were36:59:5（v/v/v）; the detection wavelength was210nm, the flow rate was1mL·min^-1, column temperature was set at30℃and the injection volume was20μL.（2）The aqueous solutions of methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin andsulfobutylether-β-cyclodextrin at a serial concentrations of0,10,20,40,80and160mmol·L^-1were prepared respectively. Excessive oleanolic acid solid powders were addedinto the above solution and then oscillated at25,35and45℃for3d. After that, thesamples were filtered through0.45μm millipore filter. The filtrated solusion wasmeasured according to the above method, and final oleanolic acid content was calculated.The phase solubility diagram was drawed with the concentration of β-cyclodextrinderivatives as horizontal ordinate and with the concentration of oleanolic acid as ordinate,and the supramolecular inclusion constants and thermodynamic parameters in the processof clusion of oleanolic acid and different derivatives of β-cyclodextrin was calculated. Thethermodynamic stability was studied. The appropriate conditions for the inclusion processof oleanolic acid and hydroxypropyl-β-cyclodextrin were screened.（3） A LC-MS methodhas been developed and validated for the determination of oleanolic acid in rat plasma.The analyte was separated on an Agilent Zorbax Eclipse Plus C18（150mm×2.1mm,5μm） column with mobile phase composed of methanol and10mmol/L ammonium acetate （90:10, v/v）. The flow rate was0.4mL·min^-1and column oven temperature was35℃.The injection volume was5μL. Glycyrrhetinic acid was used as internal standard. Massspectrometric detection was performed in electrospray positive ionization using multiplereaction monitoring （MRM）. The mass transition was m/z455.7for oleanolic acid, andm/z469.7for IS, respectively. The fragmentor voltages were180V （oleanolic acid） and200V （IS）, respectively.（4） Rats were randomly divided into the two groups，theintravenous group and oral group. The oral group was administered with oleanolicacid/hydroxypropyl-β-cyclodextrin inclusion complex by gavage, and the intravenousgroup was administered with oleanolic acid/hydroxypropyl-β-cyclodextrin inclusioncomplex by tail vein injection. Blood samples （1mL） were collected at0（pre-dose）,10,20,30,45,60,120,240and360min post dose by orbital venous plexus for oral group andat0（pre-dose）,2,5,10,20,30,60,120,240and360min post dose for intravenous group.The samples were centrifuged and the plasma was separated. The plasma oleanolic acidconcentrations were determined by LC-MS method developed in this study. The dataanalysis of pharmacokinetic parameters was performed by using Drug and Statisticsoftware （Version2.0, Chinese）. The absolute bioavailability was obtained by calculation.Results:（1） Interference test results showed a good separation of oleanolic acid andother ingredients and negative control had no significant effect on the determination ofoleanolic acid. The linear range was0.0625²mg·mL^-1. The value of precision （RSD） was0.59%, indicating a good precision for instrument. The determination of the stabilityshowed that the sample solution is stable within24h. The recoveries of oleanolicacid/methyl-β-cyclodextrin, oleanolic acid/hydroxypropyl-β-ringthe dextrin and oleanolicacid/sulfobutyl ether-β-cyclodextrin inclusion were0.66%,1.0%and1.0%, respectively.（2） As the temperature increased, the phase solubility curve slope of oleanolicacid/methyl-β-cyclodextrin was increased, but the slope of oleanolic acid/hydroxypropyl-β-cyclodextrin and oleanolic acid/sulfobutyl ether-β-cyclodextrin were decreased; At25,35and45℃, the supramolecular inclusion constants of oleanolicacid/methyl-β-cyclodextrin inclusion were6.916,10.39and27.80L·mol^-1, and thethermodynamic parameter ΔH⁰was54.59kJ·mol^-1, ΔS⁰was198.4J·mol^-1·K^-1, ΔG⁰were -4.555,-6.538and-8.522kJ·mol^-1; the supramolecular inclusion constants of oleanolicacid/hydroxypropyl-β-cyclodextrin inclusion were10.58,8.733and6.160L·mol^-1, and thethermodynamic parameter ΔH⁰was-21.24kJ·mol^-1, ΔS⁰was-51.41J·mol^-1·K^-1, ΔG⁰were-5.917,-5.403and-4.889kJ·mol^-1; the supramolecular inclusion constants of oleanolicacid/sulfobutyl ether-β-cyclodextrin were5.530,3.353and2.805L·mol^-1, and thethermodynamic parameter ΔH⁰was-26.89kJ·mol^-1, ΔS⁰was-76.36J·mol^-1·K^-1, ΔG⁰were-4.119,-3.356and-2.592kJ·mol^-1.（3） The LC-MS method for oleanolic aciddetermination in rat plasma has been developed and validated. The methodologyvalidation results showed that the extraction recoveries were good （>82%）, the lowestlimit of quantification was0.05μg·mL^-1, and the accuracy and intra-day precision weregood （RSD<15%）, indicating that the method was sensitive and specific with no matrixeffects. Above all, The method was sufficient to study the pharmacokinetics of oleanolicacid after oral administration.（4）The mean residence time of oleanolic acid in rats afteroral administration of oleanolic acid/hydroxypropyl β-cyclodextrin inclusion was84min,the peak time was at45min, and the absolute bioavailability was2.010%.Conclusion:（1） The HPLC method has been developed and validated for thedetermination of oleanolic acid in different oleanolic acid β-cyclodextrin derivativesinclusion compound. The methodology results indicate that the method meets themeasurement requirements.（2） For the first time, we studied the driving force in theinclusion process of oleanolic acid with methyl-β-cyclodextrin, hydroxypropylβ-cyclodextrin and sulfobutyl ether β-cyclodextrin. The inclusion process of oleanolic acidwith three β-cyclodextrin derivatives were spontaneous process, The inclusion process ofoleanolic acid with methyl-β-cyclodextrin was endothermic and entropy driven processwith increased disorder; The inclusion process of oleanolic acid with hydroxypropylβ-cyclodextrin and sulfobutyl ether-β-cyclodextrin were exothermic and enthalpy drivenprocess with reduced disorder.（3） The established LC-MS method for determination ofoleanolic acid in rat plasma was in line with the requirements for biological samplesdetermination.（4） As an adjunct, hydroxypropyl β-cyclodextrin could effectively improvethe bioavailability of oleanolic acid through forming inclusion with oleanolic acid.