Studies On Chemical Constituents Of Radix Glehniae Before And After Compatibility With Ophiopogon Japonicus In Vitro And Vivo

Radix Glehniae(Beishashen), from the roots of Glehnia littoralis(G. littoralis) Fr. Schmidt ex Miq.(Umbelliferae), is used to treat respiratory and gastrointestinal disorders in China. Previous study found that Radix Glehniae contained a large number of coumarins which are mainly active constituents. Ophiopogon japonicus(Maidong), from the tuberous roots of Ophiopogon japonicus(L. f.) Ker-Gawl(Liliaceae), is considered effective in treating a wide range of disorders, mainly thrombosis, myocardial ischemia, arrhythmias, respiratory disease and hyperglycemia. Previous phytochemical investigations have revealed that steroidal saponins and homoisoflavonoids are effective constituents in Ophiopogon japonicus. Radix Glehniae-Ophiopogon japonicus, a famous herb pair, is an ancient and classic formula comprised of Radix Glehniae and Ophiopogon japonicus in a weight ratio of 1:1, which has been used for treatment of mull, dryness-heat and lung and gastric yin. Up to now, there has still been no reference for chemical constituents changes before and after herb pair compatibility in vitro and vivo. Hence, in this study, sensitive and selective HPLC-ESI-MS/MS, UPLC/q TOF-MS method were developed to study on chemical constituents of Radix Glehniae before and after compatibility with Ophiopogon japonicus in vitro and vivo.Part one Simultaneous determination and comparative analysis of 19 components in Radix Glehniae, Ophiopogon japonicus and Radix Glehniae-Ophiopogon japonicus herb pair by high performance liquid chromatography-electrospray ionization tandem mass spectrometryObjective: To establish a sensitive and selective HPLC-ESI-MS/MS method for quantitative and comparative analysis of 19 components before and after compatibility between Radix Glehniae and Ophiopogon japonicus.Methods: Chromatographic conditions: Agilent Zorbax SB C18 column(4.6mm×150mm, 5μm) with a Security Guard HPLC C18 column(Agilent).; the column temperature: 25℃; mobile phase acetonitrile(A)–0.3‰ acetic acid water(B); gradient elution; flowing rate: 1 m L/min; Injection volumn: 10 μL. The electrospray ionization source was operated between positive and negative modes in a single run by multiple-reaction monitoring(MRM) modes using the following conditions: ion spray voltage, 5500 and-4500 V; the turbo spray temperature, 650 °C; curtain gas(CUR), 25 psi and interface heater was turned on; nebulizer gas(gas 1), 40 psi; heater gas(gas 2), 50 psi. The precursor-to-product ion pairs of 19 analytes were scopoletin(m/z 193.1/133.1), umbelliferone(m/z 163.0/107.1), xanthotoxol(m/z 203.1/147.1), marmesin(m/z 247.3/175.1), oxypeucedanin hydrate(m/z 305.2/203.1), psoralen(m/z 187.0/131.1), xanthotoxin(m/z 217.1/174.1), ophiopojaponin C(m/z 851.5/393.4), ophiopogon Ra(m/z 719.5/393.3), isoimpinellin(m/z 247.2/217.1), bergapten(m/z 217.1/202.1), oxypeucedanin(m/z 287.1/203.1), ophiopogonin D(m/z 855.7/287.2), imperatorin(m/z 271.1/203.0), methylophiopogonone A(m/z 341.1/219.1), mehylophiopogonanone A(m/z 343.1/135.0), methylophiopogonanone B(m/z 329.1/121.1), isoimperatorin(m/z 271.1/203.0) and ophiopogonanone C(m/z 354.9/192.7).Results: The linearity, stability, limit of detection and limit of quantification of the method were good for the 19 components. The linearity relationships﹥0.9991, instrument precision was less than 1.9 %, The intra-day and inter-day precision for 11 analytes were less than 3.8 % and 3.6 %, respectively. The recoveries were between 97.8~104.4 %. The results demonstrated that compatibility between Radix Glehniae and Ophiopogon japonicus had great effect on the contents of the main active components. The contents of coumarins decreased, while steroidal saponins and homoisoflavonoids increased after compatibility.Conclusions: A sensitive and selective HPLC-ESI-MS/MS method was developed and validated to simultaneously determinate 19 consituents in Radix Glehniae, Ophiopogon japonicus and Radix Glehniae-Ophiopogon japonicus herb pair. The method plays an important role in the study of quantitative comparison between single herb and herb pair.Part two Identification of coumarins, steroidal saponins and homoisoflavonoids in Radix Glehniae, Ophiopogon japonicus and Radix Glehniae-Ophiopogon japonicus herb pair by UPLC/q TOF-MSObjective: To summarize fragmentation rules and develop a high sensitive and efficient UPLC/q TOF-MS method for characterization and comparison of coumarins, steroidal saponins and homoisoflavonoids in Radix Glehniae, Ophiopogon japonicus and Radix Glehniae-Ophiopogon japonicus herb pair.Methods: Mixture standards, blank solution, Radix Glehniae decoction, Ophiopogon japonicus decoction and Radix Glehniae-Ophiopogon japonicus decoction were analyzed by UPLC/q TOF-MS in the positive and negative ion mode in the scanning mode of TOF-MS-IDA-8MS/MS to acquire the full-scan chromatogram. And then we could summarize fragmentation rules of the standards. The data acquisition and processing analysis were conducted using the "Master View" analysis function of Peak View 2.0 data processing software. The chemical constituents of Radix Glehniae decoction, Ophiopogon japonicus decoction and Radix Glehniae-Ophiopogon japonicus decoction were identifid by the comparison of compound retention time, precise molecular mass, elemental composition, isotopic abundance and mass spectrogram. Chromatographic conditions: Phenomenex Kinetex 2.6 μ C18 100A(50×3.0 mm,2.6 μm); the column temperature: 25℃; mobile phase 0.03% formic acid water(A)-acetonitrile(B), gradient elution; flowing rate: 0.4 m L/min. Injection volumn: 2 μL.Results: 48 coumarins in Radix Glehniae, 11 steroidal saponins and 27 homoisoflavonoids in Ophiopogon japonicus, 87 compounds(44 coumarins, 11 steroidal saponins and 32 homoisoflavonoids)in Radix Glehniae-Ophiopogon japonicus were identified and 15 differences were summarized by UPLC/q TOF-MS. 22 of these compounds were characterized by comparing with the standards, and the other 71 compounds were identified by analyzing the information with elemental composition, isotopic abundance and mass spectrogram. The error tolerance is ± 5 ppm.Conclusions: The method is simple, rapid, and high sensitivity, which can be used for qualitative and comparative analysis of the bioactive components in single herb and herb pair. 8 coumarins were identified in Radix Glehniae, while could not identified in herb pair. 3 coumarins and 4 homoisoflavonoids were identified in herb pair, while could not identified in Radix Glehniae and Ophiopogon japonicus. The method provided a new way to study the material basis of TCM herb pair.Part three Comparative Identification of the absorbed constituents and metabolites in rats after gavage administration of Radix Glehniae extract, Ophiopogon japonicus extract and Radix Glehniae-Ophiopogon japonicus extract by UPLC/q TOF-MSObjective: To develop a high sensitive and efficient UPLC/q TOF-MS method for identification the metabolites of Radix Glehniae extract, Ophiopogon japonicus extract and Radix Glehniae-Ophiopogon japonicus extract in urine, plasma and bile of rats to find the difference between single herb and herb pair and explore metabolic pathway of these herbs in vivo.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 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. The preparation of Ophiopogon japonicus extract and Radix Glehniae-Ophiopogon japonicus(1:1) extract for gavage was the same as Radix Glehniae extract. 2 Sample collection and pretreatment: 12 healthy male Sprague-Dawley rats(250±10 g) were divided into four groups randomly. 3 rats in Blank group, 3 rats in Radix Glehniae group, 3 rats in Ophiopogon japonicus group, 3 rats in Radix Glehniae-Ophiopogon japonicus group. After a single gavage administration of Radix Glehniae extract(1 m L/ 100 g), urine samples were collected 72 h, blood samples were collected at 0.08、0.17、0.33、0.5、1、2、4、6、8、10、12 and 24 h, bile samples were collected 24 h. All urine samples, plasma samples and bile samples were combined into one sample individually. All the combined urine and bile samples were extracted with triple volume of ethyl acetate. The upper solution was collected and then dried under reduced pressure. The residuary was dissolved in methanol. The plasma samples were precipitated with triple volume of acetonitrile. After centrifugation at 10000 rpm/min for 10 min, the upper solution was collected and then dried under nitrogen. The residuary was dissolved in 50 % acetonitrile. 3 UPLC/q TOF-MS conditions: Analysis was performed on a UPLC/q TOF-MS. The chromatographic separation was carried out on Phenomenex Kinetex 2.6 μ C18 100A(50×3.0 mm,2.6 μm) column; The mobile phase consisted of 0.03% formic acid water(A)-acetonitrile(B) with a gradient elution; The high-resolution mass spectrometer was operated in the positive and negative 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: The first step was to build an information database of the compounds in Radix Glehniae and Ophiopogon japonicus according to literatures. The second step was to find the main absorb components in samples by Peak View 2.0 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, steroidal saponins and homoisoflavonoids and the polarity of the compounds.Summrize and compare the metabolites whether to change or not between single herb and herb pair.Results: Scopoletin, isoimpinellin, marmesin, nodakenin, imperatorin, oxypeucedanin hydrate, ophiopogonin D’, methylophiopogonone A and ophiopogonanone E were found out as the typical compounds. Through the analysis of these typical compounds by Metabolitepilot 1.5 software, 231 main absorbed constituents and metabolites were tentatively characterized in urine, bile and plasma samples. And the possible metabolite pathway was oxidation, hydrogenation, internal hydrolysis, desaturation, demethylation, and methylation. Meanwhile, we found that there were 136(62 coumarins, 33 steroidal saponins and 41 homoisoflavonoids) different absorbed constituents and metabolites before and after compatibility between Radix Glehniae and Ophiopogon japonicus.Conclusions: The main metabolites of Radix Glehniae, Ophiopogon japonicus and Radix Glehniae-Ophiopogon japonicus herb pair in vivo were successfully screened and indentified by UPLC/q TOF-MS, which will be helpful to further study the interaction metabolism of herb pair in vivo.