| Rhodiola rosea L.belongs to the family of perennial herbaceous plant or subshruid wild plant.Due to the promising and diverse health benifits to human,Rhodiola gains its popularity in China for a long time.Specifically,its dried roots and rhizomes accounting for the major biological activities of Rhodiola has been applied widespreadly as a safe medication for centries.Pharmacological investigations on Rhodiola to date has revealed that this herb seems to be highly effective in preventing and treating a wide range of diseases including hypoxia,fatigue,aging,radiation damage,myocardial ischemia and inflammation.The chemical composition of Rhodiola has been reported to be complex,primarily phenolic glycosides,flavonoids,coumarins,amino acids and other ingredients.Among them,phenolic glycosides(salidroside)and flavonoids take major amounts and are regarded as ingredients with dominating pharmacological activities.As the only biomarker for the quality and quantity control of Rhodiola rosea defined by the Chinese Pharmacopia(edition 2010),salidroside attracts extensive interest from researchers for its potential theraputic use.The pharmacokinetic behavior of salidroside has been explored using suitable analytical method for a better understanding of its pharmacological activity,mechanism of action and clinical application.The commonly used methods for salidroside quantification include high performance liquid chromatography(HPLC),liquid chromatography-tandem mass spectrometry(LC-MS/MS)and ultra-liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Although recent studies indicate that Rhodiola flavonoids,primarily rhodiosin and rhodionin,also demonstrate remarkable pharmacological activities,the quantity and quality of investigations on their pharmacokinetics are rare.Moreover,as a potential candidate of anti-hypoxic drug,the biological fate including absorption,distribution,metabolism and excretion of Rhodiola rosea under hypoxia conditions warrants futher study.First of all,an UPLC-MS/MS method with fairly enough sensitivity and specificity was developed and validated aiming to determine the amount of salidroside and flavonoids(rhodiosin,rhodionin,herbacetin,quercetin and kaempferol)in Rhodiola rosea extract in vitro.On basis of findings from which,bioactive ingredients of Rhodiola rosea with major amounts were selected and further pharmacokinetic studies of them under hypoxia and normoxia was conducted.Since outcomes of above in vitro study identified salidroside as the major component in Rhodiola with highest amount followed by rhodiosin and rhodionin,the current study was proposed to describe and compare the pharmacokinetics of these three components in rats after administration of Rhodiola extract using UPLC-MS/MS method.The study scheme of current project was proposed as follow: 1.Quantification of six active ingredients in Rhodiola extractIn this chapter,a rapid,sensitive and specific UPLC-MS/MS method was developed to quantify six active ingredients in Rhodiola extract including salidroside and the flavonoids(rhodiosin,rhodionin,herbacetin,quercetin and kaempferol).chromatographic conditions were set as follow: Acetonitrile and 0.2 % formic acid were used as mobile phase.Chromatographic separation was performed using gradient elution with a flow rate of 0.4 ml/min.The total running time was 7 min.Mass conditions: The electrospray ionization(ESI)source was set in negative mode.The multiple reaction monitoring(MRM)analysis was conducted by monitoring the precursor ion to production transitions,including m/z 345.0?299.0 for salidroside,m/z 609.1?301.0 for rhodiosin,m/z 447.1? 301.0 for rhodionin,m/z 301.0?254.8 for herbacetin,m/z 284.8?92.9 for kaempferol and m/z 301.1?151.1 for quercetin.The precision,stability,repeatability and recovery of six analytes in current assay were fully validated.It was found that the established UPLC-MS/MS method could be successful applied to the quantification of six active ingredients in Rhodiola extract.After determination of three batches of samples,salidroside was identified as most abundance ingredient in Rhodiola extracts with a weight percentage of 1.11 %.Another two components with priority amounts in Rhodiola extracts were rhodiosin and rhodionin,with a weight percentage of 0.41 % and 0.32 %,respectively.Quercetin was found in Rhodiola extracts with only trace amount.The predominant amount of salidroside,rhodiosin and rhodionin in Rhodiola extracts,together with their promising health beneficial effects,make them popular in the field of pharmacological studies.In view of these,the pharmacokinetics of above three components were explored in the following Chapter.2.Development and validation of a UPLC-MS/MS method for determinationof salidroside,rhodiosin and rhodionin in rat plasma.A sensitive and rapid UPLC-MS/MS method for simultaneous determination of salidroside,rhodiosin and rhodionin in rat plasma has been firstly developed and validated.Puerarin was used as internal standard(IS),with the precursor ion to production transitions of 417.3?297.1.The plasma samples were prepared by methanol precipitation.The specificity of all analytes and IS in current assay met requirements from FDA and no endogenous interference from biological matrix was found.The calibration curve of salidroside,rhodiosin and rhodionin was linear over the range of 5-2500 ng/ml,10-2000 ng/ml and 10-1000 ng/ml,respectively.The lower limit of quantification(LLOQ)of salidroside,rhodiosin and rhodionin was 5 ng/ml,10 ng/ml and 10 ng/ml,respectively.The extraction recovery of three analytes at their quality control(QC)concentrations in rat plasma ranged from 63.8 % to 98.6 %.The RSD of both intra-day and inter-day precision of salidroside,rhodiosin and rhodionin were below 10.4 %,8.05 %,8.69 %,respectively;their counterpart accuracies were within the range of 91.8-100 %,89.5-97.6 % and 83.8-87.8 %,respectively.After store in the auto-sample(4?C)for 4 h,three freeze–thaw cycles and store in-80?C for 7 days,the percentages of salidroside,rhodiosin and rhodionin remaining were 88.3–109 %,76.6–99.6 % and 75.6–98.6 % in plasma,respectively.It was noticed that the recovery and stability of rhodiosin and rhodionin was generally lower than that of salidroside.This lower extraction recovery might be attributed to the nature of their structure as flavonoid glycosides with multi-hydroxyl groups which were highly susceptible to oxidation.Hence,plasma sample from rats administered with Rhodiola extract must be pretreated to improve its stability in the process of storage or sample extraction.Our preliminary study demonstrated that rhodiosin and rhodionin were stable in pH 2 to 3 plasma samples adjusted by addition of formic acid.In addition,the controlled lower pH values of biological samples could increase the extraction recovery of rhodiosin and rhodionin.3.Comparison of the pharmcokinetic charateristics of salidroside,rhodiosin and rhodionin in rat plasma under the normorxic and hypoxic conditions.Twenty male Sprague Dawley(SD)rats were randomly divided into hypoxia group(HYP)and normoxia group(NOR)with ten rats in each group.Rhodiola extract was then administered to both rats in HYP group and NOR group by oral and by intravenous to form four subgroups(n=5)including the hypoxic rats with oral administration of Rhodiola group(HYPPO),the hypoxic rats with intravenous administration of Rhodiola group(HYPIV),the normoxic rats with oral administration of Rhodiola group(NORPO),and the normoxic rats with intravenous administration of Rhodiola group(NORIV).The rats in normoxia group were placed outdoors.The rats in hypoxia group were exposed to hypoxia for 3 d before administration.Rhodiola extract was given to rats by intragastrical approach at a dose of 400 mg/kg containing salidroside 4.44 mg/kg,rhodiosin 1.64 mg/kg and rhodionin 1.28 mg/kg.As to intravenous group,Rhodiola extract was given to rats at a dose of 40 mg/kg containing salidroside 0.444 mg/kg,rhodiosin 0.164 mg/kg and rhodionin 0.128 mg/kg.The plasma concentrations of salidroside,rhodionin and rhodiosin were monitored using UPLC-MS/MS.The plasma concentration versus time profiles of each analyte in rats was analyzed by DAS software.The obtained data was analyzed by SPSS 17.0 software.A p value less than 0.05 was considered to be of statistical significance.The results showed that pharmacokinetic characteristics of salidroside,rhodiosin and rhodionin were significantly changed under hypoxia condition.It was noted that compared with that in NORPO group,AUC(0?24h)of salidroside in HYPPO group was significantly increase(p(27)0.05).The MRT(0?24h)and t1/2 of salidroside in HYPPO group were also significantly prolonged(p(27)0.001 and p(27)0.05),indicating a slower elimination compared with that in NORPO group.The Cmax and tmax values of salidroside in NORPO group and HYPPO group were similar,suggesting a comparable absorption process under two conditions.Since the mean Cmax value of rhodiosin and rhodionin in NORPO group were lower than their LLOQ in current assay(10 ng/ml),it was infeasible to draw complete plasma concentration-time curves and to calculate the pharmacokinetic parameters for these two compounds in NORPO group.The systemic exposure of rhodiosin and rhodionin in rats were also significantly increased under hypoxia condition with detectable plasma concentrations.After a single intravenous(i.v.)administration of Rhodiola extract,it was noted that compared with those in NORIV group,AUC(0?8h)of salidroside,rhodiosin and rhodionin in HYPIV group were significantly increased(p(27)0.05).The CL of salidroside,rhodiosin and rhodionin were significantly reduced(p(27)0.05).In addition,the MRT and t1/2 of rhodiosin were significantly prolonged(p<0.01),and the t1/2 of rhodionin was also prolonged(p<0.05).To summarize,the systemic exposure of these three compounds in rats was enhanced and the elimination of them was reduced under hypoxia condition.4.The pharmacokinetics of salidroside,rhodionin and rhodiosin in rats after oral administration of Rhodiola extract with 5-times doseAbove findings demonstrated that after oral administration of Rhodiola extract at 400 mg/kg,the plasma concentration of rhodionin and rhodiosin in rat was undetectable and it was infeasible to draw a complete plasma concentration vs.time curve.In the current Chapter,the oral dose of Rhodiola extract was escalated to 2000 mg/kg after a series of preliminary trials,aiming to obtain an intact plasma concentration profiles for rhodionin and rhodiosin.It was found that,in accompany with the dose escalation,the absorption phase of salidroside turned to be biphasic,which might be ascribed to its potential enterohepatic circulation or multiple absorption sites in the gastrointestinal tract.The completed plasma profiles of rhodiosin and rhodionin was obtained with a fairly low Cmax of 187.0 ng/ml and 126.0 ng/ml,respectively.The systemic exposure of rhodiosin and rhodionin in rats was rather limited with a bioavailability of 0.98 % and 1.47 %,respectively.Such low bioavailability of them,may in turn,gave an explanation for the undetectable plasma concentration of rhodiosin and rhodionin after oral administration of Rhodiola extract at low dose. |
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