A rapid and sensitive UHPLC-MS/MS method for the determination of ziyuglycoside I and its application in a preliminary pharmacokinetic study in healthy and leukopenic rats.

Ziyuglycoside I (ZgI), one of the main active ingredients in the popular Diyushengbai tablet made from Sanguisorba officinalis L., has been proven to relieve leukopenia clinically. However, to our knowledge, no studies have investigated the pharmacokinetics of either Diyushengbai tablet or ZgI in leukopenic vs. healthy individuals. In the present study, a rapid and sensitive UHPLC-MS/MS method was developed for detecting ZgI. On using this method on a novel cyclophosphamide-induced leukopenia model, we investigated differences in the pharmacokinetic characteristics of ZgI between leukopenic and normal rats. Chromatographic separation of ZgI and glycyrrhetinic acid (IS) was achieved via gradient elution in 0.5 min, and the total run time lasted for 5 min. Methodological validation results presented a good accuracy (102.6 %-110.8 %) and precision (% RSD ≤ 13.8) with a limit of quantitation of 0.5 ng/mL. Pharmacokinetic results showed a significantly shortened peak time (Tmax) (0.93 vs. 0.33 h) while a remarkably decreased maximum concentration (Cmax) (7.96 vs. 3.40 ng/L) in the 20 mg/kg leukopenia group in comparison with those in the 20 mg/kg normal group. In addition, a prolonged elimination half-life (t1/2ß) was observed in the 20 mg/kg leukopenia group (5.02 vs. 18.51 h). We observed similar trends in the 5 mg/kg oral dosing treatment and control groups, except for Cmax, which did not differ between the groups. We did not find pharmacokinetic differences in ZgI between the two leukopenia groups. Thus, the pharmacokinetic parameters of ZgI (e.g., Tmax, Cmax, and T1/2ß) changed based on the presence of a leukopenic state. This study may provide guidance for the development of ZgI as an agent for the treatment of leukopenia.

Identification and characterization of human UDP-glucuronosyltransferases responsible for the glucuronidation of fraxetin.

Fraxetin, a major constituent of the traditional medicine plant Fraxinus rhynchophylla Hance (Oleaceae), has been found to possess multiple bioactivities. However, the metabolic pathway(s) of fraxetin in human tissues has not been reported yet. This study aimed to characterize the glucuronidation pathway(s) of fraxetin in human tissues. Fraxetin could be metabolized to two glucuronides in human liver microsomes (HLMs). These two glucuronides were biosynthesized and characterized as 7-O-glucuronide (7-O-G) and 8-O-glucuronide (8-O-G). UGT1A1, -1A6, -1A7, -1A8, -1A9 and -1A10 participated in the formation of 7-O-G, while the formation of 8-O-G was catalyzed selectively by UGT1A6 and UGT1A9. UGT1A9 showed the highest catalytic activities in the formation of 7-O-G and 8-O-G. Both kinetic characterization and inhibition assays demonstrated that UGT1A9 played important roles in fraxetin glucuronidations in HLMs, especially in the formation of the major metabolite 8-O-G. Furthermore, the intrinsic clearance of fraxetin in both human liver microsomes and UGT1A9 was greater than that of 7,8-dihydroxylcoumarin, revealing that the addition of a C-6 methoxy group led to the higher metabolic clearance. In summary, the glucuronidation pathways of fraxetin in human liver microsomes were well-characterized, and UGT1A9 was the major isoform responsible for the glucuronidations of fraxetin.

Investigation of UDP-glucuronosyltransferases (UGTs) inhibitory properties of carvacrol.

UDP-glucuronosyltransferases (UGTs), the most important phase II drug metabolizing enzymes (DMEs), could metabolize many drugs and various endogenous substances including bilirubin, steroid hormones, thyroid hormones, bile acids and fat-soluble vitamins. Evaluation of the inhibitory effects of compounds on UGTs is clinically important because inhibition of UGT isoforms could not only result in serious drug-drug interactions (DDIs), but also induce metabolic disorders of endogenous substances. The aim of the present study was to investigate the inhibitory effects of carvacrol on major UGT isoforms. The results showed that carvacrol could inhibit the activity of UGT1A9 with negligible effects on other UGT isoforms. When 4-methylumbelliferone (4-MU) was used as a nonspecific probe substrate and recombinant UGT enzymes were utilized as an enzyme resource, the inhibition of UGT1A9 was best fit to the competitive type and the inhibition kinetic parameter (K(i)) was calculated to be 5.7 µM. Furthermore, another specific probe substrate, propofol, was employed to determine the inhibitory kinetics of UGT1A9, and the results demonstrated that the inhibitory type was noncompetitive. The inhibition kinetic parameter (K(i)) was determined to be 25.0 µM. Because this substrate-dependent inhibition of UGT1A9 might confuse the in vitro-in vivo extrapolation, these in vitro inhibition kinetic parameters should be interpreted with special caution.

Comparative metabolism of cinobufagin in liver microsomes from mouse, rat, dog, minipig, monkey, and human.

Cinobufagin (CB), a major bioactive component of the traditional Chinese medicine Chansu, has been reported to have potent antitumor activity. In this study, in vitro metabolism of CB among species was compared with respect to metabolic profiles, enzymes involved, and catalytic efficiency by using liver microsomes from human (HLM), mouse (MLM), rat (RLM), dog (DLM), minipig (PLM), and monkey (CyLM). Significant species differences in CB metabolism were revealed. In particular, species-specific deacetylation and epimerization combined with hydroxylation existed in RLM, whereas hydroxylation was a major pathway in HLM, MLM, DLM, PLM, and CyLM. Two monohydroxylated metabolites of CB in human and animal species were identified as 1α-hydroxylcinobufagin and 5ß-hydroxylcinobufagin by using liquid chromatography-mass spectrometry and two-dimensional NMR techniques. CYP3A4 was identified as the main isoform involved in CB hydroxylation in HLM on the basis of the chemical inhibition studies and screen assays with recombinant human cytochrome P450s. Furthermore, ketoconazole, a specific inhibitor of CYP3A, strongly inhibited CB hydroxylation in MLM, DLM, PLM, and CyLM, indicating that CYP3A was responsible for CB hydroxylation in these animal species. The apparent substrate affinity and catalytic efficiency for 1α- and 5ß-hydroxylation of CB in liver microsomes from various species were also determined. PLM appears to have K(m) and total intrinsic clearance value (V(max)/K(m)) similar to those for HLM, and the total microsomal intrinsic clearance values for CB obeyed the following order: mouse > dog > monkey > human > minipig. These findings provide vital information to better understand the metabolic behaviors of CB among various species.

Identification of cytochrome P450 (CYP) isoforms involved in the metabolism of corynoline, and assessment of its herb-drug interactions.

Corynoline, an isoquinoline alkaloid isolated from the genus Corydalis, has been demonstrated to show multiple pharmacological effects including inhibition of acetylcholinesterase, inhibition of cell adhesion, fungitoxic and cytotoxic activity. The present study focused on its metabolism and metabolism-based herb-drug interactions. After corynoline was incubated with human liver microsomes (HLMs) in the presence of NADPH, two metabolites (M-1 and M-2) were formed. Chemical inhibition experiments and assays with recombinant CYP isoforms showed that CYP2C9 was mainly involved in the formation of M-1 and CYP3A4 mainly catalysed the production of M-2. Among seven major CYP isoforms tested, corynoline showed strong inhibitory effects on the activities of CYP3A4 and CYP2C9, with an IC(50) of 3.3 ± 0.9 µm and 31.5 ± 0.5 µm, respectively. Kinetic analysis showed that inhibition of CYP3A4 by corynoline was best fit to a noncompetitive manner with K(i) of 3.2 µm, while inhibition of CYP2C9 by corynoline was best fit to a competitive manner with K(i) of 6.3 µm. Additionally, corynoline exhibited time-dependent inhibition (TDI) toward CYP3A4. The inactivation kinetic parameters (K(I) and k(inact) ) were calculated to be 6.8 µm and 0.07 min(-1) , respectively. These data are of significance for the application of corynoline and corynoline-containing herbs.

Rapid qualitative and quantitative determination of seven valuable taxanes from various Taxus species by UFLC-ESI-MS and UFLC-DAD.

The distribution and level of yew constituents vary with species and tissues. In this study, a rapid and valid method incorporating ultra-fast liquid chromatography (UFLC) with MS and UV detection was developed for simultaneous determination of paclitaxel and its six semisynthesis precursors in needles and hair roots from various Taxus species. All target analytes could be identified by comparing their retention times as well as UV and MS spectra with authentic standards, while seven valuable taxanes in botanical samples can be rapidly determined by UFLC-DAD with excellent sensitivity. Analysis of more than one hundred yew samples from nine species showed significant variations in distribution and content of seven evaluated taxanes. Thus, different developmental schemes should be used for better utilization of various yew resources.

Rapid profiling and target analysis of principal components in Fuling Decoctions by UFLC-DAD-ESI-MS.

The traditional Chinese medicine formula Fuling Decoction (FD) has been clinically used for eczema treatment, but the unclear chemical distribution and the lack of quality control have strongly restricted its application. In this study, an analytical method incorporating ultra-fast liquid chromatography (UFLC) with MS and UV detection was developed for rapid profiling of the chemical constitutes from FD. Fourteen constitutes were identified by UFLC-ESI-MS, while four major components including genipingentiobioside, geniposide, paeoniflorin and liquiritin were quantified simultaneously by UFLC-DAD. The UFLC-based method was fully validated and can be applied to screening and determination of principal components in commercially FD prescriptions.