Studies On The Coumarins And Its Metabolites In Vitro And In Vivo Of Radix Glehniae, And Study On The Quality Control Of Kangnaoshuai Capsule

Radix Glehniae (Beishashen), from the roots of Glehnia littoralis (G. littoralis) Fr. Schmidt ex Miq. (Umbelliferae), is one of the commonly used traditional Chinese medicines. The Chinese Pharmacopoeia recorded that it is always used to treat respiratory and gastrointestinal disorders in China. Previous study found that Radix Glehniae contained lots of coumarins which are active constituents. In the present dtudy, four parts of studies on Radix Glehniae were performed. (1) A new high performance capillary electrophoresis (HPCE) method was developed for the determination of 5 coumarins in Radix Glehniae and was an alternative method to quality control of Radix Glehniae. (2) A high performance liquid chromatography-mass spectrometry (HPLC-MS) method was established for the quantification of 9 coumarins in rat urine and bile after oral administration of Radix Glehniae extract and used for the excretion study. (3) The microbial transformation of isoimperatorin was studied and the transformation products were isolated and identified. (4) A high sensitive and efficient LC-MS method was developed for detection and characterization of metabolites in rat urine, feces, bile, and plasma after oral administration of isoimperatorin, and the transformation products of isoimperatorin from microbial transformation.Kangnaoshuai capsule (KNS) is a combined herbal prescription consisted of 19 herbs that are Radix Polygoni Multiflori, Radix rehmanniae preparata, Fructus lycii, Radix Ginseng, Radix Astragali, Rhizoma Dioscoreae, Radix Salviae Miltiorrhiae, Radix Paeoniae Alba, Rhizome Acori Talarinowii, radix polygalae, Semen ziziphi spinosae, Rhizoma Cyperi, Radix Scutellariae, Radix codonopsis, Indian bread with host wood, Radix ophiopogonis, Radix Puerariae and Flos Chrysanthemi. It has been used for the treatment of neurolysis, insomnia and dreamful sleep, neurastheria and memory deterioration. In the present study, a rapid and sensitive HPLC-MS method was established for the determination of 40 effective constituents. The developed method can be effectively applied for the quality control and evaluation of kangnaoshuai capsule of different production batches.Part one Simultaneous determination of 5 coumarins in Radix Glehniae by micellar electrokinetic capillary chromatographyObjective: To develop a micellar electrokinetic capillary chromatography method with ultraviolet detection for the simultaneous determination of psoralen, xanthotoxin, isoimpinellin, bergapten and scopoletin in Radix Glehniae.Methods: The separation was performed on an uncoated fused silica capillary column (50.2 cm×75μm×40 cm) with 20 mmol/L borax solution (pH 9.6) containing 16 mmol/L sodium dodecylsulfate (SDS) and 15% acetonitrile as running buffer at applied voltage of 22 kV. The detection wavelength was 214 nm. The effects of concentrations of borax solution, SDS, and organic modifier, voltage, temperature on the separation and sensitivity were investigated. The developed method was applied for the determination of 5 constituents in 16 batches of Radix Glehniae.Results: The five active constituents were completely separated within 7 min. The linear ranges of psoralen, xanthotoxin, isoimpinellin, bergapten and scopoletin were 9.91~82.6, 37.2~162, 2.23~18.6, 2.73~22.3 and 2.89~20.1μg/mL, respectively. And the average recoveries were 98.9%, 98.4%, 101.3%, 99.1% and 98.0%, respectively.Conclusion: This simple, rapid, sensitive and accurate method provided a new basis for assessment on quality of Radix Glehniae.Part two Simultaneous quantitative analysis of 9 coumarins in rat urine and bile after oral administration of Radix Glehniae extract by HPLC-MS and its application to excretion studyObjective: To develop a sensitive and selective high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of 9 coumarins in rat urine and bile after oral administration of Radix Glehniae extract. The 9 coumarins are scopoletin, xanthotoxol, xanthotoxin, psoralen, isoimpinellin, bergapten, oxypeucedanin, imperatorin and isoimperatorin.Methods: Urine and bile samples were collected after single oral administration of Radix Glehniae extract (10 mL/kg) to rat. The pimpinellin was used as internal standard (IS). The urine and bile samples were pretreated by liquid-liquid extraction with ethyl acetate (EtOAc). The chromatographic separation was performed on a C18 column with 1 mmol/L ammonium acetate and methanol as the mobile phase with gradient elution. The detection of analytes was performed on a tandem mass system equipped with a turbo ion spray interface in positive mode using multiple-reaction monitoring (MRM). The optimized mass transition ion-pairs (m/z) for quantitation were 193.1/133.0 for scopoletin, 203.2/147.1 for xanthotoxol, 217.2/202.0 for xanthotoxin, 187.2/131.1 for psoralen, 247.1/217.0 for isoimpinellin, 217.1/202.0 for bergapten, 287.2/203.1 for oxypeucedanin, 271.1/203.2 for imperatorin, 271.1/203.2 for isoimperatorin, and 247.1/231.1 for IS.Results: The calibration curves were linear over the investigated concentration range, with all correlation coefficients higher than 0.9982. The intra- and inter-day RSD were no more than 8.7% and the relative errors were within the range of -9.4% to 7.1%. The average extraction recoveries for all compounds were between 85.2-110.0%. The excretion study showed that 9 coumarins were excreted completely within 60 h in urine, while within 16 h in bile, and less than 15% of 9 coumarins were excreted in the form of prototype.Conclusion: This method was robust, specific and sensitive and it could successfully fulfill the requirement of excretion study of the 9 coumarins in Radix Glehniae.Part three Study on the microbial transformation of isoimperatorinObjective: To establish the microbial transformation method of isoimperatorin and to isolate and identify its transformation products.Methods: 15 species of fungal strains were screened and the one with the strong convert ability was selected for the transformation of isoimperatorin. The products were isolated with chromatography using silica gel and were purified with preparative liquid chromatography. Their structures were elucidated with LC-MS and NMR.Results: Cunninghamella blakesleana (AS 3.970) was found to convert isoimperatorin efficiently. Four pure products were obtained, they were oxypeucedanin hydrate, E-5-(4-hydroxy-3-methylbutyl-2-alkenyloxy)- psoralen, Z-5-(4-hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen and pranferol.Conclusion: 4 products were obtained for the first time from the microbial transformation of isoimperatorin. And among them E-5-(4-hydroxy-3-methylbutyl-2-alkenyloxy)-psoralen is a new compound.Part four Identification of in vivo metabolites and microbial transformation products of isoimperatorin by HPLC-MS and their relationships study.Objective: To develop a HPLC-MS method for detection and characterization of metabolites in rat urine, feces, bile, and plasma after oral administration of isoimperatorin, as well as the transformation products of isoimperatorin from microbial transformation.Methods: The urine, feces, bile and plasma samples were collected after oral administration of isoimperatorin to rats; and transformation solution was gathered by transformation of isoimperatorin with Cunninghamella blakesleana. The metabolites and transformation products were characterized by the combination use of the enhanced MS-information-dependent acquisition-enhanced product ion (EMS-IDA-EPI) mode, multiple ion monitoring-information-dependent acquisition-enhanced product ion (MIM-IDA-EPI) mode and precursor scan-information-dependent acquisition-enhanced product ion (PREC-IDA-EPI) mode on a hybrid triple quadrupole-linear ion trap mass spectrometer. Based on the simultaneous appearance of [M+H]+ and [M+Na]+ in the spectrum of EMS, [M+H]+ and [M+NH4]+ in the spectrum of PREC, the molecular weight of the unknown metabolites could be identified. In order to ensure the results accurate, the time of flight mass spectrometry (TOF-MS) was used to further determine the exact mass of the unknown metabolites. The structures of compounds were identified by the fragment ions. The chromatographic separation was performed on a C18 column with 1 mmol/L ammonium acetate and methanol as the mobile phase.Results: According to the fragmentation rules of furocoumarins and the comparison of reference standards, 19 metabolites were characterized in rat after oral administration of isoimperatorin, and 13 products were identified in the sample from Cunninghamella blakesleana (AS 3.970) transformation.Conclusion: In this study, a high sensitive, accurate and effective LC-MS method for qualitative analysis of the metabolites of isoimperatorin has been developed. This study demonstrated the metabolic profiling of isoimperatorin in vivo and in transformation had good correlation. The unprecedented advantage of the combination use of the EMS-IDA-EPI, MIM-IDA-EPI and PREC-IDA-EPI modes were also displayed which could easily and accurately characterize the metabolites in complex system. Part five Study on the quality control of Kangnaoshuai capsuleObjective: To establish a HPLC-ESI-MS/MS method for the simultaneous analysis of 40 constituents in Kangnaoshuai (KNS) capsule.Methods: The optimal chromatographic conditions were achieved on an Agilent C18 column with a gradient elution consisted of methanol and 0.1% formic acid in water. The precursor and product ions of analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer equipped with a turbo ion spray interface in positive and negative mode respectively using multiple–reaction monitoring (MRM). The developed method was applied for the determination of 10 bacthes of KNS capsule.Results: All calibration curves showed good linear regression (r2≥0.9986) within test ranges. The instrument, intra–day and inter–day precisions (RSD) of the investigated components were less than 1.4, 1.8 and 2.8%, respectively. And the recovery was in the range of 96.5~110.0% with RSD less than 3.3%. Conclusion: The developed method was sensitive, accurate and selective and could be applied for simultaneous determination of the major chemical components in KNS capsule. It provided an effective method for the quality control of KNS capsule.