| Salidroside and its metabolite p-tyrosol are two major phenols in Genus Rhodiola and have been confirmed possessing various pharmacological properties including resisting anoxia,anti-fatiguing,anti-aging,anti-cancer,anti-inflammation,antioxidative,hepatoprotective and cardioprotective effects and was used to the prophylaxis and therapeutics of many diseases.In this paper,the salidroside metabolite profile in rat urine was investigated,and subsequently the metabolic pathways of salidroside were proposed.After administrations of salidroside at an oral dose of 100 or 500 mg/kg,rat urine samples were collected and pretreated with methanol to precipitate the proteins.The pretreated samples were analyzed by an Acquity ultraperformance liquid chromatography(UPLC)coupled with an HSS T3 column and detected by quadrupole time of-flight mass spectrometry(Q-TOF-MS)or high-performance liquid chromatography coupled with hybrid triple-quadrupole linear ion trap mass spectrometry(HPLC/Q-trap-MS).A total of eight metabolites were detected and identified on the basis of the characteristics of their protonated ions in the urine samples.The results elucidated that salidroside was metabolized via glucuronidation,sulfation,deglycosylation,hydroxylation,methylation,and dehydroxylation pathways in vivo.we optimized determination method of salidroside and p-tyrosol in rat tissue,excretion and intestinal absorption samples,and made a systemic method validation.The tissue distribution,excretion and intestinal absorption of salidroside and p-tyrosol in rats were investigated after oral administration at a dose of 100 mg/kg in order to the metabolism regularity.The uncompleted absorption and drugs can get into water by urine,feces and bile to harm biology and environment.So we simulate different temperture and pH value for the hydrolysis studies and different optical wavelength and light intensity for the photolysis studies of salidrisode,to provide important effect on understanding the behavior of salidroside in environment deeply.In selected spectral condition,the compounds can be completely separation,and the determination of salidroside and p-tyrosol can't be interfered by internal compounds;the LLOD of both were 50 ng/mL,and the LOD value was 20 ng/mL;The regression equation fate of standard curve was 0.99,and the linear range was 50-2000 ng/mL,the results of precision and accuracy indicated that the method have better repeatability,and satisfied the condition of quantitative determination.An UPLC-MS/MS method was developed to quantitatively determine salidroside and p-tyrosol in rat tissue,feces,urine and bile samples.The method was simple,quickly,accurate and precise,selectivity enough and has been successfully applied to the pharmacokinetic study.The tissue distribution regulation of salidroside and p-tyrosol was investigated after i.p.administration at a dose of 100 mg/kg,the results indicated that the content of salidroside was only detected in liver,the contents of 1 h and 2 h after administration were significantly higher than others which were 1113.07 ± 244.31 ng/g and 1088.87±165.66 ng/g,respectively,and then the concentration of salidroside in liver decreased quickly,almost completely cleared until 4 h after administration.A significant amount of p-tyrosol was detected in kidney,followed by liver and heart,the content of spleen was significantly lower then other tissues 1 h after administration,and also almost completely cleared until 4 h after administration.After i.p.administration at a dose of 100 mg/kg,about 23.80%salidroside was excretion by urine,the cumulative contents of salidroside in urine was 4337.10±766.43?g until 72 h.Then,bile was another main excretion route,and the cumulative contents of salidroside was 4.65±0.13?g which was 0.02%of dosage.There was no salidroside in feces.There was little p-tyrosol in urine,the total cumulative was 407.10 ± 37.08?g,which was 2.35%of dosage.The total cumulative of salidroside and p-tyrosol was 26.17%of dosage.The intestinal absorption characteristics of salidroside and p-tyrosol were studied by in vivo intestinal perfusion technology.The results indicated that there was absorption difference in different parts of intestinal,the absorption of jejunum was significant higher than others,the ABS%of jejunum was 15.62%,there was no significant difference in Pw*during all four parts of intestinal,the percent of accumulation absorption of salidroside during 1.5 h-2 h was the maximum which was 32.19±3.12%,K value order was jejunum<colonic<duodenum<ileum,and the opposite order in T1/2,the value were 7.68±2.09 d?6.17±0.16 d?2.29±0.13 d and 1.76±0.34d,respectively;The absorption ofp-tyrosol in four parts of intestinal were have no significant difference,ABS%was between 23.81%and 32.81%,the percent of accumulation absorption of p-tyrosol was between 30.43±7.68%?42.96±5.85%,and both of them were higher than that of salidroside.The hydrolysis of salidroside was promoted by temperature,hydrolysis rate will be faster as along as the higher of temperature and the hydrolysis rates at 35? and 45 ? were consistent with the first-order kinetic equation.The hydrolyzed rate constant and the half-life period were 0.07 and 5.92 d at 35? and the hydrolyzed rate constant and the half-life period were 0.117 and 10.04 d at 45?;The hydrolysis of salidroside can be promoted significantly by weak acid in the water environment,however,there was no salidroside to be hydrolyzed in pH 8.0;The hydrolysis rate in pH 5.4 and pH 7.4 were consistent with the first-order kinetic equation.The hydrolyzed rate constant and the half-life period in pH 5.4 were 0.12 and 5.29 d,respectively,but the hydrolyzed rate constant and the half-life period in pH 7.4 were 0.062 and 11.18 d,respectivelyThe salidroside solution were exposure to ultraviolet lamp with dominant wavelength at 254,308 and 365 nm,the results showed that the photolysis regularity of salidroside in different wavelength was not consistent with the first-order kinetic equation.The photolysis degree was as follow:254 nm>308 nm>365 nm,so the photolysis degree was profitable with short wavelength of radiation.Salidroside aqueous solution was exposed by simulated sunlight irradiation with 1800,4800,7200 and 11000 Lx,respectively.The photolysis degree of salidroside was corresponding with increased light intensity gradually.But when after 48 h,the photolysis degree of salidroside did not increase with increased prolonged exposure time,the maximum photolysis rate was 35%,the rest of the drug can't be decomposed by the light effect.And the photolysis process of salidroside affected by light intensity also was not consistent with the first-order kinetic equation. |
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