Studies On The Pharmacokinetics And Metabolism Of Carnosic Acid In Rats

Rosemary(Rosmarinus officinalis L.) and sage(Salvia officinalis L.) have been widely used as natural antioxidants,due to their very high antioxidant activity.Carnosic acid(CA) is a phenolic diterpene,which is considered to be the most important antioxidant constituent of rosemary and sage,and its content represents a quality parameter for such products.In addition to its antioxidant activity,CA also shows anti-inflammatory,antiplatelet,antibacterial, anticancer,antivirus,and photo-protective activities.For the purpose of investigating and clarifying the physiological disposition of CA and promoting its rational application, systematical study on the absorption,distribution,metabolism,and excretion(ADME) of CA in plasma or other biological fluids were proceeded.In-vitro microbial transformation model and rat liver microsome incubation model were also established for predicting the metabolism of CA.The metabolic pathways of CA on these two different models were compared and the correlation between the in vitro and in vivo process of CA were evaluated.The results were as follows:1.A simple and effective HPLC method with ultraviolet detection was firstly established for the quantification of CA in rat plasma.The intra-day and inter-day relative standard deviations(RSD) for the measurements of quality control samples were less than 2.7%and 3.1%,respectively.The recovery for plasma samples was≥72.2%.The stability of the plasma samples was also validated.The validated results of methodology were in accordance with relevant regulations,and all data and results showed that the HPLC method could be applied to determine the concentration of CA in rat plasma precisely.The assay was successfully applied to the pharmacokinetic study and the absolute bioavailability assessing of CA in rat after different dosage of intragastric(60,90 and 120 mg·kg^-1) and intravenous(1,5 and 10 mg.kg^-1) administration,respectively.The absorption of CA was slow after intragastric administration.However,the maximum plasma concentration was high and retained for a long time.The absolute bioavailability of CA was more than 60%, the average value was 63.29±1.60%,which would be a useful feature in future clinical applications of the drug as an antioxidant.2.A HPLC method was established to determine the concentration of CA in tissues.This method was quick,precise,and reproducible,and could be used to quantify prototype in rat liver,stomach and small intestine after intragastric administration(90 mg·kg^-1).It was the first time to study the tissue distribution of CA in rats after intragastric administration.The result indicated that after intragastric administration CA distributed in stomach,liver and small intestine with the C_max of 1871.3,16.13 and 34.19μg.g^-1 respectively,but it was not detected in other tissues,including high blood flow tissues such as heart,kidney and lung.3.In order to clarify why the concentration of CA was high in rat plasma and very low in rat tissues,an equilibrium dialysis method was established to determine the concentration of CA in rat plasma and dialysis solutions.This method was successfully applied to study the plasma-protein bonding rate of CA.The result showed that the plasma-protein bonding rate of CA was among 61.9-87.7%,which indicated that the phenomenon mentioned above was the result of high plasma-protein bonding rate.4.Simple and effective HPLC methods with ultraviolet detection were established for the quantification of CA in rat bile,urine and feces,respectively.These methods were quick, precise,and reproducible.The assays were successfully applied to the excretion study of CA prototype in rat bile,urine and feces,respectively.The result indicated that after intragastric administration(90 mg.kg^-1),the cumulative excretion amount of CA in bile,urine,and feces were 5.086,2.787 and 1499.6μg,respectively.The feces were proved to be the main excretion path of CA in rat and 7.272%prototype was excreted from this excretion path. While there were still about 92.7%of CA was proposed to be metabolized and excreted as the metabolites of CA.This indicates that CA is extensively metabolized in rat.5.The metabolites of CA in rat bile,urine,and feces were identified by RRLC-MSⁿ.4 metabolites were detected in rat bile,and among them 1 was analyzed,15 metabolites were detected in rat urine,and 13 were identified among them.3 metabolites were detected and identified in rat feces,which indicated that CA was extensively metabolized in rat.The types of metabolite reaction were varied,among them,the oxidation,reduction and conjugation reactions were main metabolite types.The metabolism paths of CA in rats were tentatively proposed on the base of metabolites identification results.6.The in-vitro microbial transformation model was used to study the in vitro metabolites of CA,and 3 metabolites were identified using RRLC-MSⁿ.In-vitro rat liver microsome incubation model was established,using this model 2 metabolites were detected and identified by RRLC-MSⁿ.The metabolic pathways of CA on these two different models were compared and the correlation of in-vitro and in-vivo metabolites of CA was also studied.