ß-Glucuronidase- and OATP2B1-mediated drug interaction of scutellarin in Dengzhan Xixin Injection: A formulation aspect.

Scutellarin is the major and active constituent of Dengzhan Xixin Injection (DZXX), a traditional Chinese medicine prepared from the aqueous extract of Erigeron breviscapus and widely used for the treatment of various cerebrovascular diseases in clinic. In present study, the possible pharmacokinetic differences of scutellarin after intravenous administration of scutellarin alone or DZXX were explored. Additional, the potential roles of ß-glucuronidase (GLU) and OATP2B1 in drug-drug interaction (DDI) between scutellarin and constituents of DZXX were further evaluated in vitro. The plasma concentration, urinary and biliary excretion of scutellarin in rats after administration of DZXX, were significantly higher than those received scutellarin, while pharmacokinetic profile of Apigenin 7-O-glucuronide (AG) in rats was similar no matter AG or DZXX group. Furthermore, higher concentration in brain and plasma, however, lower level of scutellarin in intestine were observed after intravenous administration of DZXX. Finally, AG and caffeoylquinic acid esters were found to significantly inhibit GLU and OATP2B1 in vitro, which might explain, at least in part, the pharmacokinetic DDI between scutellarin and other chemical constituents in DZXX. The findings provided deep insight into the prescription-formulating principle in DZXX for treating the cerebrovascular diseases.

Mechanistic studies on the absorption and disposition of scutellarin in humans: selective OATP2B1-mediated hepatic uptake is a likely key determinant for its unique pharmacokinetic characteristics.

Scutellarin [scutellarein-7-O-glucuronide (S-7-G)] displayed a unique pharmacokinetic profile in humans after oral administration: the original compound was hardly detected, whereas its isomeric metabolite isoscutellarin [scutellarein-6-O-glucuronide (S-6-G)] had a markedly high exposure. Previous rat study revealed that S-7-G and S-6-G in the blood mainly originated from their aglycone in enterocytes, and that the S-7-G/S-6-G ratio declined dramatically because of a higher hepatic elimination of S-7-G. In the present study, metabolite profiling in human excreta demonstrated that the major metabolic pathway for S-6-G and S-7-G was through further glucuronidation. To further understand the cause for the exposure difference between S-7-G and S-6-G in humans, studies were conducted to uncover mechanisms underlying their formation and elimination. In vitro metabolism study suggested that S-7-G was formed more easily but metabolized more slowly in human intestinal and hepatic microsomes. Efflux transporter study showed that S-6-G and S-7-G were good substrates of breast cancer resistance protein and multidrug resistance-associated protein (MRP) 2 and possible substrates of MRP3; however, there was no preference great enough to alter the S-7-G/S-6-G ratio in the blood. Among the major hepatic anion uptake transporters, organic anion-transporting polypeptide (OATP) 2B1 played a predominant role in the hepatic uptake of S-6-G and S-7-G and showed greater preference for S-7-G with higher affinity than S-6-G (K(m) values were 1.77 and 43.9 µM, respectively). Considering the low intrinsic permeability of S-6-G and S-7-G and the role of OATP2B1 in the hepatic clearance of such compounds, the selective hepatic uptake of S-7-G mediated by OATP2B1 is likely a key determinant for the much lower systemic exposure of S-7-G than S-6-G in humans.

Use of hollow fiber liquid phase microextraction and HPLC for extraction and determination of apigenin in human urine after consumption of Satureja sahendica Bornm.

The applicability of hollow fiber liquid phase microextraction (HF-LPME) was evaluated for extraction and preconcentration of apigenin prior to its determination by HPLC. Different parameters affecting the HF-LPME recovery such as nature of organic solvent, pH of donor and acceptor phases, extraction time, stirring speed, salt addition were optimized. Under optimum conditions (1-octanol as organic solvent, pH of the donor phase=3 and pH of acceptor phase=11.5, extraction time of 75 min, stirring speed of 1000 rpm) limit of detection (LOD) of 0.1 ng/mL, linear range of 0.5-300 ng/mL and correlation of determination (R(2)) of 0.9956 were obtained. The relative intra and inter-day standard deviations (RSD%) based on five replicate measurement were 3.5% and 10.7% respectively. Enrichment factor of 315 and recovery 85% were achieved. Finally, the applicability of the proposed method was evaluated by extraction and determination of apigenin in urine sample after consumption of Satureja sahendica Bornm. which is a native medicinal plant from Iran. Concentration of apigenin in urine sample was found to be 6.20 ng/mL.

Absorption and disposition of scutellarin in rats: a pharmacokinetic explanation for the high exposure of its isomeric metabolite.

Scutellarin or scutellarein-7-O-glucuronide (S-7-G) is a flavonoid used in the treatment of cardiovascular diseases. After oral administration to humans, S-7-G can hardly be detected, whereas its isomeric metabolite [scutellarein-6-O-glucuronide (S-6-G)] dominates in plasma. A preliminary study in rats also revealed a low bioavailability of S-7-G, as well as a high plasma concentration of S-6-G. Therefore, the present study tried to explore the possible causes of the unusual pharmacokinetics of scutellarin in humans through investigating the absorption and disposition of S-7-G in rats. After oral administration to rats, S-7-G was largely hydrolyzed in the intestinal tract and was absorbed as aglycone. While passing through the intestinal wall, aglycone was extensively glucuronidated into S-7-G and S-6-G (approximately 20:1), which subsequently entered the mesenteric blood (approximately 15:1). However, because S-7-G exhibited more rapid uptake in hepatocytes, was glucuronidated at a 2.7-fold higher rate in the liver, and was excreted in greater amounts through bile and urine than S-6-G, the S-7-G/S-6-G ratio eventually declined to approximately 1.5:1 in the systemic circulation. Findings revealed that S-7-G cannot be absorbed directly; S-7-G and S-6-G in the body were mostly generated from aglycone in the intestinal wall; a larger amount of S-7-G than S-6-G entered the mesenteric blood at the absorption stage, but the gap between them shrank quickly mainly because of the higher hepatic first-pass elimination of S-7-G. These findings in rats are of great value as reference for further study to accurately interpret the pharmacokinetics of S-7-G in humans.

Metabolism profile of scutellarin in urine following oral administration to rats by ultra performance liquid chromatography coupled to time-of-flight mass spectrometry.

Scutellarin, a flavone glucuronide of 5,6,4'-trihydroxyflavone-7-O-glucoronide, is the main active component of the traditional Chinese botanic drug Erigeron breviscapus (Vant.) Hand.-Mazz. In this study, a method based on ultra performance liquid chromatography coupled with a time-of-flight mass spectrometer (UPLC/TOF MS) was established and validated to profile the metabolites of scutellarin in Sprague-Dawley rat urine following oral administration of single dose of scutellarin at 80.8 mg/kg. The column utilized was an Acquity BEH C18 (150 mm x 2.1 mm, 1.7 microm). The mobile phase was 0.2% formic acid and acetonitrile with gradient condition. Two standard curves of scutellarin were obtained for the concentration range of 1.065-10.65 microg/mL and 10.65-63.92 microg/mL, respectively. By automating the data processing of the software Masslynx developed by Waters Ltd., 17 metabolites of scutellarin were found and determined in rat urine, with the corresponding reactions in vivo such as isomerism, reduction, methylation, glucuronide conjugation, hydroxylation, hydroxylation and methylation, etc., most of which were discovered for the first time. For most metabolites, the time (T(p)) of peak excretion was 8-12h. Calculated as scutellarin, the cumulative urine excretion rate of the metabolites was 1.93%.

Two major urinary metabolites of scutellarin in rats.

Urinary metabolites of scutellarin, the main effective constituent of breviscapine, a cerebrovascular and cardiovascular drug consisting of total flavonoids of Erigeron breviscapus, were investigated in rats. Two major metabolites were isolated from the urine of rats following oral administration of scutellarin and identified as scutellarein 6,7-di- O-beta-D-glucuronide (M1) and scutellarein (M2), respectively, on the basis of chemical and spectroscopic evidence. M1 was reported as a metabolite of scutellarin for the first time.

Bioavailability of apigenin from apiin-rich parsley in humans.

AIM: Absorption and excretion of apigenin after the ingestion of apiin-rich food, i.e. parsley, was tested. METHODS: Eleven healthy subjects (5 women, 6 men) in the age range of 23-41 years and with an average body mass index of 23.9 +/- 4.1 kg/m2 took part in this study. After an apigenin- and luteolin-free diet, a single oral bolus of 2 g blanched parsley (corresponding to 65.8 +/- 15.5 micromol apigenin) per kilogram body weight was consumed. Blood samples were taken at 0, 4, 6, 7, 8, 9, 10, 11 and 28 h after parsley consumption and 24-hour urine samples were collected. Apigenin was analyzed in plasma, urine and red blood cells by means of HPLC-ECD. RESULTS: On average, a maximum apigenin plasma concentration of 127 +/- 81 nmol/l was reached after 7.2 +/- 1.3 h with a high range of variation between subjects. For all participants, plasma apigenin concentration rose after bolus ingestion and fell within 28 h under the detection limit (2.3 nmol/l). The average apigenin content in 24-hour urine was 144 +/- 110 nmol/24 h corresponding to 0.22 +/- 0.16% of the ingested dose. The flavone could be detected in red blood cells without showing dose-response characteristics. CONCLUSIONS: A small portion of apigenin provided by food reaches the human circulation and, therefore, may reveal biological effects.

Determination and assay validation of luteolin and apigenin in human urine after oral administration of tablet of Chrysanthemum morifolium extract by HPLC.

A simple, selective, precise, and accurate RP-HPLC assay for simultaneous analysis of luteolin and apigenin in human urine was developed and validated. Prior to HPLC analysis, urine samples were incubated with beta-glucuronidase/sulfatase. Separation and quantification were achieved on an Agilent C18 column under isocratic conditions using a mobile phase (methanol:0.2% phosphoric acid aqueous solution 55:45, v/v) maintained at 1.0 ml/min at 30 degrees C. The standard curves were linear over the range of 0.0975-7.800 and 0.1744-13.95 microg/ml for luteolin and apigenin, respectively (r > 0.999). The assay recoveries for luteolin and apigenin were above 85.7%. The intra-day and inter-day precision (R.S.D.) for luteolin were below 2.2 and 4.0%, respectively, and for apigenin were less than 2.8 and 5.4%, respectively. Stability studies showed three concentration of luteolin and apigenin in urine quality control samples were stable undergoing three freeze-thaw cycles, storage at room temperature for 4 h, and at -20 degrees C for 3 days. The limit of quantitation was 39.20 ng/ml (n = 5) for luteolin and 31.45 ng/ml (n = 5) for apigenin in human urine. The method developed was employed successfully to determine luteolin and apigenin in urine samples obtained from eight healthy volunteers following oral administration of tablet of Chrysanthemum morifolium extract (CME).

Pharmacokinetics and metabolism of apigenin in female and male rats after a single oral administration.

The metabolism of apigenin, a weak estrogenic flavonoid phytochemical, was investigated in the rat. After a single oral administration of radiolabeled apigenin, 51.0% of radioactivity was recovered in urine, 12.0% in feces, 1.2% in the blood, 0.4% in the kidneys, 9.4% in the intestine, 1.2% in the liver, and 24.8% in the rest of the body within 10 days. Sex differences appear with regard to the nature of compounds eliminated via the urinary route: immature male and female rats, like mature female rats, excreted a higher percentage of the mono-glucuronoconjugate of apigenin than the mono-sulfoconjugate of apigenin (10.0-31.6% versus 2.0-3.6%, respectively). Mature male rats excreted the same compounds in an inverse ratio (4.9% and 13.9%, respectively). Radioactivity appeared in the blood only 24 h after oral administration. Blood kinetics showed a high elimination half-time (91.8 h), a distribution volume of 259 ml, and a plasmatic clearance of 1.95 ml/h. All of the parameters calculated from these experiments suggested a slow metabolism of apigenin, with a slow absorption and a slow elimination phase. Thus, a possible accumulation of this flavonoid in the body can be hypothesized.