[Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin].

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 µL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 µL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 µL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 µL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 µL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 µL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 µL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 µL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 µL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 µL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 µL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 µL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 µL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

Deciphering the Relationship Between the Trough Concentration of Posaconazole and Its Efficacy and Safety in Chinese Patients With Hematological Disorders.

Posaconazole (PCZ) is effective in preventing and salvage treatment invasive fungal infections in patients with hematologic disorders. However, PCZ displays highly variable individual pharmacokinetics affecting its efficacy and safety. To investigate the correlation between PCZ concentration and efficacy and safety, the following key influencing factors were explored. A total of 285 trough plasma concentrations (Cmin) of 81 Chinese patients receiving PCZ oral suspension for prophylaxis or treatment of invasive fungal infections were collected in this study. The relationships between Cmin values and clinical response and hepatotoxicity were investigated as well as the incidence of clinical response under different Cmin values of PCZ with a logistic regression model. The concentration of PCZ showed remarkable differences among patients with haematologic disorders. PCZ Cmin values of 0.76 and 1.0 µg/mL were both associated with an over 80% probability of successful response to prophylaxis and treatment of fungal infections, respectively. No association between Cmin values and hepatotoxicity was noted (P > 0.05). Gender, albumin, and co-administration of proton pump inhibitor (PPI) were identified as independent factors influencing PCZ Cmin by multiple linear regression analysis. Furthermore, patients' C-reactive protein (CRP), albumin, and co-administration of PPI exhibited significant effects on the therapeutic window of patients receiving PCZ for prophylaxis. The plasma concentration is closely associated with therapeutic efficacy of PCZ. It is necessary to adjust the dosing regimens based on PCZ Cmin to obtain an optimal therapeutic response.

An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems.

The human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most essential conjugative enzymes, is responsible for the metabolism and detoxification of bilirubin and other endogenous substances, as well as many different xenobiotic compounds. Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices. Herein, an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices, using liquid chromatography with fluorescence detection (LC-FD), has been developed and validated. The newly developed LC-FD based assay has been confirmed in terms of sensitivity, specificity, precision, quantitative linear range and stability. One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate, enabling reliable quantification of lower amounts of active enzyme than any other method. The precision test demonstrated that both intra- and inter-day variations for this assay were less than 5.5%. Furthermore, the newly developed assay has also been successfully used to screen and characterize the regulatory effects of small molecules on the expression level of UGT1A1 in living cells. Overall, an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems, providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases, interactions with xenobiotics, and characterization modulatory effects of small molecules on this conjugative enzyme.

Discovery of cardio-protective constituents of Gualou Xiebai Decoction, a classical traditional Chinese medicinal formula.

BACKGROUD: Finding effective compounds of TCMs has always been the basis for achieving marker-based quality control which is currently most widely used quality control strategy. Gualou Xiebai Decoction (GLXB), a classical TCM formula, is recorded and proven as a therapy for curing coronary heart disease but the effective constituents are unidentified and the substantial basis of the therapeutic effects is not clear. PURPOSE: The present research is an investigation on the chemistry of this formula aiming at finding and precisely identifying effective compounds. STUDY DESIGN AND METHODS: This research started with screening for effective fractions of GLXB by rat myocardial infarction model and H9c2 cell hypoxia/reoxygenation model, then compounds in effective fractions were isolated and identified by phytochemical and spectroscopic methods. The cardio-protective activities of the compounds were tested in vitro and one of the effective compounds was taken as example to investigate the mechanisms. RESULTS: The water-insoluble parts of GLXB were identified as effective parts in both in vitro and in vivo experiments. Systematic isolation of compounds in the effective fractions resulted in the isolation of 34 compounds including 7 new compounds, whereas 8 compounds were effective in protecting H9c2 cells against hypoxia/reoxygenation injury. One of the effective compounds, macrostemonoside P (MP) possibly exerted its effect by activating RISK pathway and attenuating apoptosis. CONCLUSION: An array of effective constituents of GLXB were discovered, and discovery of these compounds contributed to elucidating the substantial basis for the therapeutic effects of this formula, and provides fundaments for establishing Q-markers for further reliable quality control of GLXB.

Systematic screening and characterization of Qi-Li-Qiang-Xin capsule-related xenobiotics in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Qi-Li-Qiang-Xin capsule (QLQX), a well-known traditional Chinese medicine prescription (TCMP), is consisted of eleven commonly used herbal medicines, has been widely used for the treatment of chronic heart failure (CHF). However, the absorbed components and related metabolites after oral administration of QLQX are still remaining unknown. In the present work, a reliable and effective method using ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) was established to identify QLQX-related xenobiotics in rats. Based on a representative structure based homologous xenobiotics identification (RSBHXI) strategy, a total of eleven compounds (salvianolic acid B, formononetin, benzoylmesaconine, alisol A, sinapine thiocyanate, naringin, tanshinone IIA, ginsenoside Rg1, ginsenoside Rb1, astragaloside IV and periplocin), bearing different chemical core structures, were selected and investigated for their metabolism in vivo. And then, comprehensive metabolic profiles of the holistic multi-ingredients in QLQX were achieved. As a result, a total of 121 QLQX-related xenobiotics (47 prototypes and 74 metabolites) were identified or tentatively characterized, among them eight prototypes (mesaconine, hypaconine, songorine, fuziline, neoline, talatizamine formononetin, neocryptotanshinone) and two metabolites (calycosin-gluA, formononetin-guA) were relatively the main existing xenobiotics exposed in blood. All absorbed prototype constituents were mainly from six composed herbal medicines (Aconiti lateralis radix, Astragali radix, Ginseng radix, Alismatis rhizoma, Salvia miltiorrhiza radix, Periploca cortex). The main metabolic reactions were methylation, hydrogenation, hydroxylation, oxidization, sulfation and glucuronidation. This is the first study on in vivo metabolism of QLQX. These results enabled us to focus on several high exposure ingredients in the discovery of effective substances of QLQX, however further pharmacokinetic study on these QLQX-related xenobiotics are needed to be carried out.

Simultaneous Quantification of Multiple Representative Components in the Xian-Ling-Gu-Bao Capsule by Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Xian-Ling-Gu-Bao capsule (XLGB), a famous traditional Chinese medicine prescription, is extensively used for the treatment of osteoporosis in China. However, few studies on the holistic quality control of XLGB have been reported. In this study, a reliable method using 18 representative components in XLGB was successfully established and applied to evaluate 34 batches of XLGB samples by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The choice of quantitative markers mostly followed four principles, i.e., absorbed components in plasma, bioactive compounds with in vitro anti-osteoporosis activity, those derived from multiple individual medicinal herbs in XLGB with multiple representative structure types, and quantitative chemical markers in the Chinese Pharmacopoeia. The results showed chemical consistency was good except for individual batches. Multivariate statistical analysis indicated that asperosaponin VI from Radix Dipsaci, epimedin C, magnoflorine, and icariin from Herba Epimedii as well as timosaponin BII from Rhizoma Anemarrhenae varied significantly in multiple samples, which hinted an assay for these four components should be completed during all of the manufacturing processes. Taken together, this study provided a feasible method for holistic quality control of XLGB by multiple chemical markers, which could play a vital role in guaranteeing the safety, effectiveness, and controllability of administering the capsules as a medication in clinics.

Identification, bioactivity evaluation and pharmacokinetics of multiple components in rat serum after oral administration of Xian-Ling-Gu-Bao capsule by ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

The Xian-Ling-Gu-Bao capsule (XLGB) is a famous traditional Chinese medicine prescription (TCMP), which has proven effective in osteoporosis treatment. However, due to the lack of a dynamic XLGB profile, the in vivo pharmacokinetics of multiple bioactive components within this medicine remains unknown. In the present study, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) identified a total of eighteen prototypes (using reference standards) in rat serum after oral administration of XLGB. These prototypes were subsequently evaluated to ascertain their effects on the proliferation and alkaline phosphatase activity of UMR106 cells and the adipogenesis of 3T3-L1 cells. Furthermore, a rapid and sensitive UPLC/Q-TOF-MS method was developed and validated for simultaneous quantitative analysis of 11 prototypes in rat serum. Chromatographic separation was achieved using a Waters Acquity BEH C18 column (2.1×100mm, 1.7µm) and linear gradient elution employing a mobile phase consisting of water and acetonitrile (both containing 0.1% formic acid). All calibration curves showed excellent linearity (r2>0.99) within the sampling ranges considered. The assay was accurate, precise and reproducible, as demonstrated by the obtained intra- and inter-day precisions (less than 12.3%) and accuracies (between -12.7% and 11.0%), and the matrix effects, extraction recoveries and stabilities were all satisfactory. Moreover, pharmacokinetic parameters were calculated from the plasma concentration-time data. Compared to single-compound dosing, significantly enhanced responses were obtained when several analytes were administered simultaneously, indicating possible drug-drug interactions among the complex ingredients of TCMP. This work provides an experimental baseline regarding the clinical applications and medicinal effectiveness of XLGB in the treatment of osteoporosis.

Phytochemistry and pharmacology of Allii Macrostemonis Bulbus, a traditional Chinese medicine.

Allii Macrostemonis Bulbus (Xiebai in Chinese), as a famous traditional Chinese medicine, has great medicinal and dietary values since ancient times. In China, the dry bulbs of Allium macrostemon and Allium chinense are both used as its original plants. Pharmacological studies have revealed that both of them could increase plasminogen activator activity and prolong the effect of coagulation to achieve antiplatelet aggregation which validates their traditional uses for the treatment of thoracic obstruction and cardialgia in clinics. Besides, several other significant activities, including lipid-lowering, anti-atherosclerosis, antitumor, antispasmodic, antibacterial, antioxidant, and insecticidal activities, have already been reported. The volatile oils, nitrogenous compounds, and steroidal saponins are the major beneficial compounds. Among them, steroidal saponins are considered as the characteristic constituents. In this review, the current information concerning the phytochemistry and pharmacology of Allii Macrostemonis Bulbus is summarized comprehensively. In addition, several research future perspectives are presented, especially the mechanism of bioactive components and fraction from the bulbs of Allium macrostemon and Allium chinense.

Study on chemical profiles and metabolites of Allii Macrostemonis Bulbus as well as its representative steroidal saponins in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Allii Macrostemonis Bulbus (AMB) is increasingly becoming popular as an edible vegetable or traditional folk medicine in East Asia due to its great health and medicinal properties. However, due to a lack of available research, the effective material of AMB still remains unknown. In this study, we applied a strategy utilising ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) to investigate chemical profiles of AMB. In addition, metabolite profiles of five representative single steroidal saponins as well as AMB were investigated. Moreover, the metabolic features of saponins in AMB were summarised. After oral administration, the saponins underwent massive phase I and phase II metabolism. Sequential deglycosylation metabolism in rat intestine was the main metabolic pathway of the steroidal saponins, while oxidation, dehydrogenation, glucuronic acid reactions in liver also take part in further modification. These results expand our knowledge about the metabolism of AMB.

Isolation and identification of phase I metabolites of phillyrin in rats.

Phillyrin was one of the main chemical constituents of the fruit of Forsythia suspensa (Thunb.) Vahl. It showed various bioactivities including antioxidant and anti-inflammatory activities. However, the metabolism of phillyrin remained unknown. This report described the isolation and identification of phase I metabolites of phillyrin in rats. Nine metabolites including six new ones were isolated by various column chromatographies and high-performance liquid chromatography. Their structures were elucidated by extensive spectroscopic analysis. The antiviral activities of phillyrin and the metabolites were evaluated against influenza A (H3N2) virus. Among them, one metabolite M8 showed moderate activity with the IC50 value of 26.39 µM, and three metabolites (M2, M3, M9) showed weak antiviral activities at the concentration of 100 µM. Based on the structures of the metabolites, possible metabolic pathways of phillyrin in rats were also proposed.

Metabolites profile of Xian-Ling-Gu-Bao capsule, a traditional Chinese medicine prescription, in rats by ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry analysis.

Xian-Ling-Gu-Bao capsule (XLGB), a well-known traditional Chinese medicine prescription (TCMP), is widely used for the treatment of osteoporosis. However, due to lack of metabolism research, the effective material of XLGB is still unknown. It entails a huge obstacle for the clinical-safe medication administration and quality control of XLGB. To explore the metabolic fate of multiple components of XLGB, herein, we proposed a "representative structure based homologous xenobiotics identification" (RSBHXI) strategy based on ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) and mass defect filter (MDF) technique. A total of 147 XLGB-related xenobiotics were identified or tentatively characterized in rat biofluids after oral administration of XLGB, including 134 (57 prototype components and 77 metabolites) in plasma, 93 (37 prototype components and 56 metabolites) in urine and 118 (46 prototype components and 72 metabolites) in bile. Our results indicated that prenylated flavonol glycosides from Herba epimedii, prenylated flavonoids from Fructus psoraleae, saponins from Radix dipsaci and Rhizoma anemarrahenae, as well as tanshinones from Radix Salviae Miltiorrhizae were major absorbed chemical components of XLGB. Hydrolysis, glucuronidation and sulfation were major metabolic reactions of XLGB. As more xenobiotics were detected in bile than those in urine, it demonstrated that multiple components of XLGB underwent comprehensive hepatobiliary excretion. The present study expands our knowledge about the metabolism of XLGB which will be conducive to revealing its in vivo pharmacological material basis. In addition, the application of RSBHXI strategy provides a new approach for metabolite identification of TCMPs and other complex mixture.