Preparation,pharmacokinetics And Pharmacodynamics Evaluation Of Rosmarinic Acid Phospholipid Complex And It’s Oil Solution

Rosmarinic acid（RA）is a kind of water-soluble phenolic acid compounds,is one of the main active components of chinese medicine,such as Salvia officinalis,Prunella vulgaris,Perilla frutescens,Rosmarinus officinalis.RA exhibited various pharmacological effects,including antioxidant,hepatoprotective,anti-inflammatory,anticancer and neuroprotective effects.It can play a role of a pharmacological while auxiliary therapeutic effect on other diseases,has a good application prospect.However,RA has the poor solubility,poor permeability(P_app<1 ×10^-10 cm/s)and low absolute bioavailability（0.91～1.70%）,which restricts its application.In this paper,RA-PLC were prepared by solvent-evaporation and characterized.Intestinal cellular permeability was examined using Caco-2 cell monolayers in vitro and pharmacokinetics in vivo was also undertaken.Then dissolved RA-PLC into medium-chain triacylglycerols to form RA-PLC oil solution（RA-PLC-ME）,pharmacokinetics and pharmacodynamics in vivo was also undertaken.The main contents include the preparation of RA-PLC and RA-PLC-ME.Intestinal cellular permeability was examined using Caco-2 cell monolayers in vitro and pharmacokinetics and pharmacodynamics in vivo was also undertaken.RA-PLC were prepared by solvent-evaporation,the complexing rate of RA and phospholipid as an index,the reaction solvent,the concentration of RA,the molar ratio of RA/phospholipid,reaction time and reaction temperature were examed.On the basis of the single factor investigation,we select the concentration of reactants,weight ratio of phospholipids and RA（w/w）,and reaction time,which influence notable of complexing rate,and then a central composite design approach was employed for process optimization.The best preparing conditions:the reaction solvent was anhydrous ethanol,the reactant concentration was 1.5 mg/mL,the ratio of RA/phospholipid was 1:2,the reaction time was 2 h and the reaction temperature was 50℃.The best preparation conditions were verified,and the results showed that the repeatability was good.RA-PLC were characterized by powder X-ray diffraction（XRD）,Fourier transform infrared（FTIR）spectroscopy,and differential scanning calorimetry（DSC）.Additionally,oil/water partition coefficients（P）were determined.As showed in DSC and XRD curve,the crystalline peaks of RA has disappeared in the RA-PLC,the RA-PLC exists in the form of amorphous.The FTIR spectra showed that in the physical mixture of RA and the phospholipid（PM）,the characteristic peaks of RA still exist.This suggested that there was no interaction between RA and the phospholipid.However,these characteristic peaks disappeared in RA-PLC.These changes were probably attributable to RA and the phospholipid forming a complex through hydrogen bonding between the-OH group of the phenol rings of RA and the P=O group of the phospholipid.The n-octanol/water partition coefficients（P）showed that in comparison to unformulated RA,the complex exhibited an increase in the P value,which resulted from an increased partitioning into the organic phase,improved lipophilicity and/or the amorphous nature of RA within the RA-PLC complex.Compared with unformulated RA,the P of PM was slightly increased,possibly because the phospholipids slightly improved the hydrophilicity of RA in physical mixture by means of its solubilization.Intestinal cellular permeability of RA and RA-PLC were examined using Caco-2 cell monolayers in vitro.The transport result show that the P_app of RA and RA-PLC from the apical（AP）to basal（BL）side,were（0.86±0.12）× 10^-10 cm/s and（2.71±0.89）× 10^-10 cm/s,respectively;From the basal（BL）to apical（AP）side,were（0.71±10.05）×10^-10 cm/s and（1.98±0.73）× 10^-10 cm/s,respectively.Compared with RA,RA-PLC significantly increase the P_app of RA（p<0.05）.It suggested that RA-PLC increased the membrane permeability of RA.The efflux ratio of RA and RA-PLC were both low than 1.5,This result indicated that the RA might transport across Caco-2 monolayer via passive diffusion.Establish accurate,reliable and easy UPLC-MS/MS method for determination of the RA content in plasma of rats and validate the methodology.rats were randomly divided into different groups（i,e.unformulated RA group and RA-PLC group）,each group was administered at a dose level equivalent to 50 mg of RA/kg p.o.The blood concentration was tested by UPLC-MS/MS.Compared with unformulated RA,RA-PLC presented higher A UC_0-t,and C_max（p<0.05）.The C_max and AUC_0-t of RA-PLC were 1.47-and 1.2-fold higher,respectively,compared to unformulated RA.It is evident that RA-PLC exhibited higher absorption and relative bioavailability than unformulated RA.Pharmacokinetic result of RA-PLC-ME showed that,C_max and AUC_0-t of RA-PLC-ME were 3.25-and 2.91-fold higher respectively,compared to unformulated RA;Compared with RA-PLC,RA-PLC-ME presented higher AUC_0-t and C_max（p<0.01）,were 2.21-and 2.42-fold higher respectively.These resulets showed that,RA-PLC-ME could further enhance oral bioavailability of RA.The antioxidant and hepatoprotection effects of unformulated RA and RA-PLC-ME on acute hepatic injury induced by CCL₄ in mice were also studyed.The result showed that both the unformulated RA and RA-PLC-ME treated groups had reducing the increase in hepatosomatic index,alanine aminotransferase（ALT）and aspartate aminotransferase（AST）,elevated superoxide dismutase（SOD）,catalase（CAT）,glutathione peroxidase（GSH-Px）and the levels of malondialdehyde（MDA）.Compared with unformulated RA,RA-PLC-ME showed better hepatoprotection effects on liver injury induced by CCL₄ in mice.The all results showed that,compared with RA-PLC,RA-PLC-ME could further improved lipophilicity,membrane permeability,enhance oral bioavailability,antioxidant and hepatoprotection effects of RA.