Identification Of The Metabolites In Rats After Gavage Administration Of Radix Glehniae Extract And The Study On The Interaction Between Radix Glehniae And Radix Ophiopogonis In Pharmacokinetics

Radix Glehniae(Beishashen), from the roots of Glehnia littoralis(G. littoralis) Fr. Schmidt ex Miq.(Umbelliferae), is one of the most commonly used traditional Chinese medicines(TCM). It has been recorded in Chinese Pharmacopoeia and is used to treat respiratory and gastrointestinal disorders in China. Previous study found that Radix Glehniae contained a large number of coumarins which are active constituents. Up to date, there is no study to systematically characterize the metabolites of coumarins in Radix Glehniae. The metabolites of TCM not only are more complicated than TCM themselves but also have trace levels in vivo. Therefore, it is critical to accurately and rapidly identify the constituents or metabolites of TCM in vivo in order to get a better understanding about TCM. UPLC/q TOF-MS possesses high sensitivity and good separation efficacy, as well as provides the elemental compositions based on the accurate mass measurement, which makes it widely used for the qualitative and quantitative analysis in TCM and the metabolites in vivo. In this study, UPLC/q TOF-MS is employed to identify the metabolites of Radix Glehniae extract in urine, plasma and bile of rats and then the possible metabolite pathway will be deduced. Besides, a strategy will be established to find the metabolites of TCM in vivo.Radix Glehniae and Radix Ophiopogonis are famous TCM, and also used together in clinic for hundreds of years to nourish yin and remove heat for they supply each other in curative effect. However, the mechanism of the interaction between them is still unclear. In the present study, UPLC/q TOF-MS was performed to characterize the pharmacokinetics of the main constituents in rats after administration of Radix Glehniae, Radix Ophiopogonis, Radix Glehniae-Radix Ophiopogonis, which lay the foundation for the study of the mechanism of the interaction between them. Part one Identification of the absorbed constituents and metabolites in ats after gavage administration of Radix Glehniae extractObjective: To establish a universal and effective strategy for finding and indentifying the metabolites of TCM in vivo by UPLC/q TOF-MS and to characterize the metabolites of Radix Glehniae extract in urine, plasma and bile of rats through this strategy.Methods:1 Preparation of Radix Glehniae extract for gavage: Radix Glehniae was decocted with boiling water according to the traditional method. The extraction solution was concentrated to a level which equivalent to 0.4 g/m L of the raw Radix Glehniae and then precipitated with quadruple volumes of dehydrated ethanol. The precipitate was discarded and the ethanol was removed under reduced pressure. The residuary solution was diluted with water to get the Radix Glehniae extract with a concentration equivalent to 2 g/m L of the Radix Glehniae material.2 Sample collection and pretreatment: 9 healthy male Sprague-Dawley rats(250±10 g) were divided into three groups(3 rats / group) randomly. After a single gavage administration of Radix Glehniae extract(1 m L/ 100 g), urine samples were collected 72 h in group 1, bile samples were collected 24 h in group 2, and blood samples were collected at 0.17、0.5、1、2、3、4、6、8、10、12 and 24 h in group 3. All urine samples, bile samples and plasma samples were combined into one sample individually. All the combined samples were extracted with ethyl acetate. The upper solution was collected and then dried under reduced pressure. The residuary was dissolved in methanol.3 UPLC/q TOF-MS conditions: Analysis was performed on a UPLC/q TOF-MS. The chromatographic separation was carried out on Kinelex 2.6 μ C18 100A(100×2.10 mm)(Phenomenex) with a Security Guard UHPLC C18 column(Phenomenex). The mobile phase consisted of methanol and 1 mmol/L aqueous ammonium acetate solution with a gradient elution. A single run was 35 min. The high-resolution mass spectrometer was operated in the positive ion electrospray mode. A full scan was employed in MS1. DBS was used to trigger the IDA function in the experiment. For the IDA criteria, the eight most intense candidate ions of per cycle that exceeded 100 cps counts were selected to do a production scan.4 Analytical strategy: For the identification of the metabolites, four steps were applied. The first step was to build an information database of the compounds in Radix Glehniae according to literatures. The second step was to find the main absorb components in samples by Peak View 1.2 software. Then classify them into several groups based on their structures, and find out a typical compound in each group. The third step was to analyze these typical compounds by Metabolitepilot 1.5 software to find out the main metabolites of the absorbed constituents in urine, bile and plasma samples. The last step was to identify or tentatively characterize the metabolites based on the cleavage pathway of coumarins and the polarity of the compounds. Besides, the possible metabolite pathway could be deduced according to the metabolites.Results: Twenty main absorbed constituents were found in the bio-samples by Peak View 1.2 software. These constituents were classified into five groups, and scopoletin, isoimpinellin, imperatorin, 6-(3,3-dimethylallyl)-7-hydroxycoumarin and marmesin were found out as the typical compounds for these groups. Through the analysis of these typical compounds by Metabolitepilot 1.5 software, 111 main absorbed constituents and metabolites were tentatively characterized in urine, bile and plasma samples. And the possible metabolite pathway was oxidation, methylation, hydrolysis, hydrogenation, sulfate conjugation and glucuronide conjugation.Conclusion: The main metabolites of Radix Glehniae in vivo were successfully screened and indentified by UPLC/q TOF-MS, which will be helpful to better understand the metabolism of Radix Glehniae in vivo. At the same time, we provide an effective method to analyze the absorb components and metabolites of TCM in vivo.Part two he study on the interaction between Radix Glehniae nd Radix Ophiopogonis in pharmacokineticsObjective: To determine the main constituents of Radix Glehniae and Radix Ophiopogonis in plasma by UPLC/q TOF-MS in order to study the interaction between Radix Glehniae and Radix Ophiopogonis in pharmacokinetics which will lay the foundation for the study of the mechanism of the interaction between them.Methods:1 Preparation of Radix Glehniae extract, Radix Ophiopogonis extract, and Radix Glehniae-Radix Ophiopogonis extract for gavage: Radix Glehniae and Radix Ophiopogonis were decocted with boiling water individually according to the traditional method. The extraction solutions of Radix Glehniae and Radix Ophiopogonis were concentrated for 8-fold and 4-fold, respectively. Then the concentrated extracts were disposed by freeze drying to prepare the powders of them. The powders of Radix Glehniae and Radix Ophiopogonis were diluted with water to prepare Radix Glehniae extract(equivalent to 1 g/m L of the Radix Glehniae material), Radix Ophiopogonis extract(equivalent to 1 g/m L of the Radix Ophiopogonis material), and Radix Glehniae-Radix Ophiopogonis extract(equivalent to 1 g/m L of the Radix Glehniae material and 1 g/m L of Radix Ophiopogonis material) for gavage.2 Sample collection and pretreatment: 18 healthy male Sprague-Dawley rats(270±20 g) were randomly divided into three groups(6 rats / group, Radix Glehniae group, Radix Ophiopogonis group, and Radix Glehniae-Radix Ophiopogonis group) and administrated of above extracts individually(2 m L/ 100 g). Blood samples were collected at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24 and 36 h from the posterior orbital veinusing in each group after a single dosing. Plasma samples were prepared with acetonitrile by simple protein precipitation.3 UPLC/q TOF-MS conditions: The chromatographic separation was carried out on an Phenomenex Kinetex 2.6 μ C18 100A(50×3.0 mm, 2.6 μm) with a column filter. The mobile phase consisted of acetonitrile(A) and 0.03% acetic acid(B) with a linear gradient elution. Electrospray ion source and product ion scan mode were employed. The detection of xanthotoxin, bergapten, isoimpinellin, imperatorin and psoralen from Radix Glehniae was under positive ion mode, while ophiopogonin D, methylophiopogonanone A, methylophiopogonanone B and methylophiopogonone A from Radix Ophiopogonis was under negative ion mode. The sum of multiple accurate product ions was used to obtain the peak area of analytes, and internal standard method and weighted(1/x2) simple linear regression were employed to determine the concerntration of the analytes in plasma samples.4 Analysis in pharmacokinetics: Plasma concentration-time profile and the related pharmacokinetic parameters of these components were obtainted through the above determination to study the interaction between Radix Glehniae or Radix Ophiopogonis group and Radix Glehniae-Radix Ophiopogonis group.Results: The linearity of xanthotoxin, bergapten, isoimpinellin, imperatorin, psoralen, ophiopogonin D, methylophiopogonanone A, methylophiopogonanone B and methylophiopogonone A in rat plasma were good in the ranges of 0.040~20 ng/m L, 0.040~20 ng/m L, 0.040~20 ng/m L, 0.06~30 ng/m L, 0.06~30 ng/m L, 0.800~200 ng/m L, 0.30~75 ng/m L, 0.30~75 ng/m L and 0.30~75 ng/m L, respectively, with r ≥ 0.991. The LLOQ of all analytes were ≤ 0.04 ng/m L. The RSD of intra- and inter-batch were ≤10.1%, and RE was in the range of-5.0% and 8.7%. The mean extraction recoveries and matrix effect of all analytes were in the ranges of 67.4 %~105.5% and 88.1%~112.5%, respectively. The coadministration of Radix Glehniae and Radix Ophiopogonis can increase the bioavailabiility of bergapten, isoimpinellin, psoralen, imperatorin, ophiopogonin D and methylophiopogonone A, change the process of bergapten, isoimpinellin and psoralen, slow down the absorption of methylophiopogonanone A and methylophiopogonanone B.Conclusion: The treatment of freezing-drying for Radix Glehniae extrat and Radix Ophiopogonis extract was benefit for prolonging their storage life. The developed UPLC/q TOF-MS method is rapid, sensitive, specific and stabile can be employed for the determination of the constituents from Radix Glehniae and Radix Ophiopogonis in plasma. The results of pharmacokinetics after coadministration of Radix Glehniae and Radix Ophiopogonis are useful for the further study of the mechanism of making prescription.