LC-MS/MS for determination of aesculetin in rat plasma and its application to a pharmacokinetic study.

Aesculetin, a coumarin compound present in the sancho tree and chicory, exhibits excellent antioxidant and anti-inflammatory activities in the vascular and immune system. In this study, a rapid and sensitive ultra-high performance liquid chromatography electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method was established and validated for the determination of aesculetin in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Chromatographic separation was performed on an Acquity UPLC HSS T3 C18 column (2.1 × 100 mm, 1.8 µm) with gradient elution at a flow rate of 0.3 ml/min, using mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). Aesculetin and puerarin (internal standard) were detected by multiple reaction monitoring in negative ion mode. The method was fully validated according to the US Food and Drug Administration guidelines. The calibration curve was linear over the range of 2-1,000 ng/ml with correlation coefficient >0.9980. The carry-over, matrix effect, extraction recovery, dilution effect, intra- and inter-day precision and the accuracy were within acceptable limits. The method was then applied to a pharmacokinetic study of aesculetin in rats. After oral administration at doses of 5, 10 and 20 mg/kg, the plasma concentration reached peaks of 95.7, 219.9, 388.6 ng/ml at times of 1.22-1.78 h. The oral bioavailability was calculated as 15.6-20.3% in rat plasma. The result provided pre-clinical information for further application of aesculetin.

Coumarin glycosides from Hydrangea paniculata slow down the progression of diabetic nephropathy by targeting Nrf2 anti-oxidation and smad2/3-mediated profibrosis.

BACKGROUND: Water extract of Hydrangea paniculata (HP) stem, rich in coumarin glycosides, has been demonstrated to have renal protective effect in several experimental kidney injury animal models. Currently, it is under pre-clinical development as a class 5 herbal drug against membranous nephropathy. However, whether it also benefits diabetic nephropathy (DN) is not clear. PURPOSE: This study was performed to investigate the protective effect of HP on streptozotocin-induced experimental DN, and further understand its molecular mechanisms. METHODS: In the present study, type 1 diabetes rat model was established by the intraperitoneal injection of streptozotocin. HP was orally administered every day for three months. Biochemical analysis and histopathological staining were conducted to evaluate the renal functions. In vivo pharmacokinetic study was conducted to analyse the metabolites of HP with high blood drug concentration. In vitro assay using these metabolites was performed to analyse their ability to reduce reactive oxygen species (ROS) production induced under high glucose (HG) condition by flow cytometry. Reverse transcription-polymerase chain reaction was conducted to analyse the mRNA level of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and IL6 and western blot was performed to analyse the phosphorylation status of smad 2/3 in HK2 cells under TGFß1 stimulation. RESULTS: The treatment with HP significantly reduced the blood urea nitrogen and serum creatinine content, and urine albumin excretion in diabetic rats, and increased the creatinine clearance rate. Periodic acid-schiff and methenamine staining and immunohistochemistry revealed that HP also ameliorated glomerulosclerosis and tubular vacuolar degeneration, as well as the deposition of fibronectin and collagen IV in the glomeruli. Pharmacokinetic study results revealed that the major coumarin compounds from HP were metabolised into umbelliferone and esculetin. By in vitro assay, umbelliferone and esculetin were found to significantly decrease ROS production induced by HG content, as well as increase the mRNA level of Nrf2. HP and its metabolites also can down-regulate fibronectin secretion in HK2 cells stimulated by TGFß1 and inhibit smad2/3 phosphorylation. CONCLUSION: HP has beneficial effect on DN by increasing Nrf2 expression and inhibiting TGF-smad signal activation. Further, it can be a novel herbal drug against DN.

Breast Cancer Resistance Protein and Multidrug Resistance Protein 2 Determine the Disposition of Esculetin-7-O-Glucuronide and 4-Methylesculetin-7-O-Glucuronide.

Esculetin (ET)-7-O-glucuronide (ET-G) and 4-methylesculetin (4-ME)-7-O-glucuronide (4-ME-G) are the main glucuronide of ET and 4-ME, respectively. The disposition mediated by efflux transporters for glucuronide has significant influence on the pharmacokinetic profile and efficacy of bioactive compounds. In the current study, transporter gene knockout mice and Caco-2 cells were used to explore the effects of breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) on the disposition of ET-G and 4-ME-G. After oral or i.v. administration of ET and 4-ME, the area under the plasma concentration-time curve from time 0 to the last data point or infinity values of ET, 4-ME, and their glucuronides (ET-G and 4-ME-G) were remarkably and significantly increased in most Bcrp1-/- and Mrp2-/- mice compared with those in wild-type FVB mice (P < 0.05). These results were accompanied with a significant increase of maximum plasma concentration values (P < 0.05). In Caco-2 monolayers, the efflux and clearance rates of ET-G and 4-ME-G were markedly reduced by the BCRP inhibitor Ko143 and MRP2 inhibitor MK571 on the apical side (P < 0.05). In an intestinal perfusion study, the excretion of ET-G was significantly decreased in perfusate and increased in plasma in Bcrp1-/- mice compared with those in wild-type FVB mice (P < 0.05). The 4-ME-G concentration was also decreased in the bile in transporter gene knockout mice. ET and 4-ME showed good permeability in both Caco-2 monolayers [apparent permeability (Papp ) ≥ 0.59 × 10-5 cm/s] and duodenum (Papp ≥ 1.81). In conclusion, BCRP and MRP2 are involved in excreting ET-G and 4-ME-G. ET and 4-ME are most likely absorbed via passive diffusion in the intestines.

Development of a daphnetin transdermal patch using chemical enhancer strategy: insights of the enhancement effect of Transcutol P and the assessment of pharmacodynamics.

OBJECTIVE: The aim of this study was to develop a drug-in-adhesive patch for transdermal delivery of daphnetin (DA), which is a coumarin derivative in Girald Daphne, and to investigate the role of Transcutol P (TP) in the release and percutaneous permeation processes of DA. METHODS: Backing films, permeation enhancers and enhancer content in the transdermal patch were investigated through in vitro experiments using rat skin. Anti-inflammatory and analgesic effects of the optimized formulation were evaluated using the adjuvant arthritis model and the pain model induced by acetic acid, respectively. In addition, the enhancement effect of TP was investigated using differential scanning calorimetry (DSC), FTIR, and molecular dynamic simulation. RESULTS: The optimal formulation, composed of DURO-TAK® 87-2852, CoTranTM 9680, 1% DA, and 10% TP showed anti-inflammatory and analgesic effects. It was found that TP only promoted the release process of DA from its transdermal patch. Furthermore, the decrease of interaction between drug and pressure sensitive adhesive (PSA) as well as the improvement of PSA mobility due to TP addition were the main factors that enhanced the release of DA from patch. CONCLUSIONS: This study successfully used TP to develop a DA patch with good anti-inflammatory and analgesic effects, proving that TP promotes the release of DA by reducing the interaction between DA and PSA and increasing the mobility of PSA.

[Simultaneous determination of daphnetin, daphnoretin, daphneticin in rat plasma by LC-MS/MS and its application in pharmacokinetic study].

To establish HPLC-MS/MS method for simultaneous determination of daphnetin, daphnoretin, and daphneticin in rat plasma after oral and intravenous administration of Daphne giraldii extract, and then use them in the calculation of pharmacokinetic parameters. Six sprague-dawley rats received intragastric administration of D. giraldii extract (daphnetin, daphnoretin and daphneticin were 88.40, 3.24 and 4.28 mg•kg⁻¹, respectively). Their drug plasma concentration was determined by LC-MS/MS with schisandrin as an internal standard to draw plasma concentration-time curve. The pharmacokinetic parameters were calculated by Kinetica 4.4. The results showed that the linear range was 5-1 000 µg•L⁻¹ for daphnetin, daphnoretin and daphneticin, and the method ological test showed conformance to the requirements.The intraday and inter-day variable coefficients (RSD) were both less than 15.0%, indicating that both of legitimate precise and accuracy were consistent with the analysis requirements of biological samples. For daphnetin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 4 h, 858.96 µg•L⁻¹, 10 566.4 µg•L⁻¹â€¢h, 5.19 h and 9.43 h, respectively. For daphnoretin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 2.92 h, 178.00 µg•L⁻¹, 905.89 µg•L⁻¹â€¢h, 3.50 h and 6.95 h, respectively. For daphneticin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 2 h, 36.67 µg•L⁻¹, 355.11 µg•L⁻¹â€¢h, 4.95 h and 8.27 h, respectively. The LC-MS/MS analysis method established in this study was proved to be so accurate and sensitive that it can be applied to the pharmacokinetic study of daphnetin, daphnoretin and daphneticin.

The interaction of auraptene and other oxyprenylated phenylpropanoids with glucose transporter type 4.

BACKGROUND: Glucose transporter 4 (GLUT4) is firmly established to play a pivotal role in glucose metabolism and in particular in modulating the insulin-stimulated glucose transport in several tissues, such as skeletal muscle and adipose tissue. Stimulation of GLUT4 by insulin results in its translocation to the plasma membrane, activation of several kinases, and finally in a large glucose influx into cells. PURPOSE: In this study we investigated the modulating properties of four biologically active oxyprenylated ferulic acid and umbelliferone derivatives and of their unprenylated parent compounds on GLUT-4 mediated glucose uptake and translocation. METHODS: Oxyprenylated phenylpropanoids have been synthesized in high yields and purity by already reported methodologies. All the synthesized chemicals were tested for their capacity to modulate GLUT4 mediated glucose uptake and GLUT4 translocation in L6 rat skeletal myoblasts in the concentration range 0.1 - 10 µM. Insulin (0.1 µM) was used as positive control. Western blot analysis was employed to assess if GLUT4 translocation occurred prior to increase of glucose uptake. Statistical analyses were carried out by the Dunnett multiple comparison test. RESULTS: 4'-Geranyloxyferulic acid (GOFA), 7-isopentenyloxycoumarin, and auraptene (7-geranyloxycoumarin) increased glucose uptake in a concentration-dependent manner, and significant increases were observed at 0.1 µM for GOFA, and 10 µM for 7-isopentenyloxycoumarin, and auraptene. These products also were able to significantly promote the translocation of GLUT4 to the plasma membrane of L6 myotubes. After treatment with compounds for 15 min, the incorporated amounts of GOFA, 7-isopentenyloxucoumarin, and auraptene were 0.15, 0.32, and 1.77 nmols/60-mm culture dish, respectively. A sample of raw Italian propolis, found to be rich in GOFA and auraptene, was also seen to mimic insulin-effect in the concentration range 0.01 - 1.0 mg/ml. CONCLUSIONS: Among the compounds assayed, auraptene showed to possess potentialities to be a potent activator of both translocation of GLUT4 and glucose influx into skeletal muscle cells with the highest bioavailability among effective compounds. Its capacity to modulate sugar metabolism, coupled to its presence in edible Citrus fruits, can be regarded as an additional reason to account for the already known stimulating properties of some vegetable (e.g. bitter orange).

Assessment of the Combined Treatment with Umbelliferone and Four Classical Antiepileptic Drugs Against Maximal Electroshock-Induced Seizures in Mice.

BACKGROUND/AIMS: The aim of this study was to determine the effects of umbelliferone (7-hydroxycoumarin; UMB) on the anticonvulsant potency of four classical antiepileptic drugs (carbamazepine (CBZ), phenytoin (PHT), phenobarbital (PB) and valproate (VPA)) in the mouse maximal electroshock-induced seizure (MES) model. RESULTS: UMB administered systemically intraperitoneally (ip) in a dose of 150 mg/kg significantly elevated the threshold for maximal electroconvulsions (p < 0.05) in mice. Moreover, UMB (150 mg/kg) co-administered with PB and VPA significantly enhanced the anticonvulsant potency of these drugs by reducing their median effective doses (ED50 values) from 35.39 to 21.78 mg/kg (p < 0.01) for PB, and from 281.4 to 215.5 mg/kg (p < 0.01) for VPA. In contrast, UMB (150 mg/kg, ip) had no significant effect on the antiseizure activity of CBZ and PHT in the mouse MES model. Neither total brain PB, nor total brain VPA concentrations were altered after ip administration of UMB, indicating a pharmacodynamic nature of interactions between the tested drugs. CONCLUSIONS: The selective potentiation of the anticonvulsant potency of PB and VPA by UMB, and lack of any pharmacokinetic interactions between drugs, make the combinations of UMB with PB or VPA worthy of consideration for epileptic patients who are refractory to standard antiepileptic treatment.

A kinetic study of the main guaco metabolites using syrup formulation and the identification of an alternative route of coumarin metabolism in humans.

For decades guaco species have been empirically used for the treatment of respiratory diseases. However, studies have shown that the toxic and therapeutic effects of the main guaco metabolites are dose-dependent, and none clinical study was done to evaluate the behavior of these substances in humans. In this work, a pilot study measuring the kinetic profile of the main guaco metabolites was performed leading to the knowledge of an alternative route of coumarin metabolism in humans. Initial screenings demonstrated that the administration of 60 mL of guaco syrup (single dose) did not provide sufficient levels of coumarin (COU), 7-hydroxycoumarin (7-HCOU), o-coumaric acid (OCA) and kaurenoic acid (KAU). The pharmacokinetic parameters were calculated by orally administering 60 mL of guaco syrup spiked with 1500 mg of COU. The kinetic study demonstrated that the plasmatic levels of 7-HCOU (considered the main metabolite of COU) were 10 times lower than the levels of COU, and the kinetic profile of 7-HCOU suggests sequential metabolism in the liver with low access of 7-HCOU to the systemic circulation. The study also demonstrated that OCA is one of the main bioavailable metabolites of COU. Therefore, the hydrolysis of the lactone ring forming a carboxylated compound is one of the possible routes of COU metabolism in humans. The half-lives of COU, 7-HCOU and OCA were approximately 4.0, 1.0 and 3.0 h, respectively and there was evidence that the recommended dosage of guaco syrup did not provide sufficient levels of COU, 7-HCOU or OCA to obtain a bronchodilation effect. Clinical studies are necessary to prove the efficacy and safety of products based on guaco.

Investigation of (E)-3-[4-(2-Oxo-3-aryl-chromen-4-yl)oxyphenyl]acrylic Acids as Oral Selective Estrogen Receptor Down-Regulators.

A novel estrogen receptor down-regulator, 7-hydroxycoumarin (5, SS5020), has been reported with antitumor effects against chemically induced mammary tumors. Here, we report on our own investigation of 7-hydroxycoumarins as potential selective estrogen receptor down-regulators, which led us to the discovery of potent down-regulating antagonists, such as 33. Subsequent optimization and removal of the 7-hydroxy group led to coumarin 59, which had increased potency and improved rat bioavailability relative to SS5020.

Structural modifications at the C-4 position strongly affect the glucuronidation of 6,7-dihydroxycoumarins.

Esculetin (6,7-dihydroxycoumarin) and its C-4 derivatives have multiple pharmacologic activities, but the poor metabolic stability of these catechols has severely restricted their application in the clinic. Glucuronidation plays important roles in catechols elimination, although thus far the effects of structural modifications on the metabolic selectivity and the catalytic efficacy of the human UDP-glucuronosyltransferase (UGT) enzymes remain unclear. This study was aimed at exploring the structure-glucuronidation relationship of esculetin and its C-4 derivatives, including 4-methyl esculetin, 4-phenyl esculetin, and 4-hydroxymethyl esculetin as well as 4-acetic acid esculetin. It was achieved by identifying the main human UGTs responsible for the different reactions and by careful characterization of the reactions kinetics. These catechols, with the exception of 4-acetic acid esculetin, are selectively metabolized to the corresponding 7-O-glucuronides. UGT1A6 and UGT1A9 are the two major UGTs involved in the 7-O-glucuronidation of 4-methyl esculetin and esculetin. UGT1A6 was the major contributor for 7-O-glucuronidation of 4-hydroxymethyl esculetin, and UGT1A9 played a significant role in the 7-O-glucuronidation of 4-phenyl esculetin. The results of the kinetic analyses revealed that the Km values of the compounds, in both UGT1A9 and human liver microsomes, decreased with increasing hydrophobicity of the C-4 substitutions. The outcome of this was that C-4 hydrophobic and hydrophilic groups on 6,7-dihydroxycoumarin exhibited contrasting effects on UGT affinity. All of these findings provide helpful guidance for further structural modification of 6,7-dihydroxycoumarins with improved metabolic stability.

Phytochemicals as inhibitors of bacterial cell division protein FtsZ: coumarins are promising candidates.

Naturally occurring phytochemicals with reported antibacterial activity were screened for their ability to inhibit the bacterial cell division protein Escherichia coli FtsZ. Among the representative compounds, coumarins inhibit the GTPase and polymerization activities of this protein effectively. Further screening with ten coumarin analogs we identified two promising candidates, scopoletin and daphnetin. The former is found to inhibit the GTPase activity of the protein in a noncompetitive manner. Docking of these coumarins with the modeled protein indicate that they bind to T7 loop, which is different from the GTP-binding site (active site), thereby supporting the experimental data. Lowest binding energy is obtained with scopoletin. 3D QSAR indicates the need for groups such as hydroxyl, diethyl, or dimethyl amino in the 7th carbon for enhanced activity. None of the coumarins exhibited cytotoxicity against NIH/3T3 and human embryonic kidney cell lines. The length of Bacillus subtilis increases in the presence of these compounds probably due to the lack of septum formation. Results of this study indicate the role of coumarins in halting the first step of bacterial cell division process.

The effects of Glycyrrhizae uralenis and its major bioactive components on pharmacokinetics of daphnetin in Cortex daphnes in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhizae uralenis (GU) is often prescribed together with Cortex daphnes (CD) in traditional Chinese medicinal practice to increase the efficacy of CD on the treatment of rheumatoid arthritis (RA), but the reasons were still unknown. In order to clarify the rationality of herbaceous compatibility between CD and GU, the comparative evaluations on pharmacokinetic behaviors of daphnetin (a predominantly active ingredient in CD) after intragastric administration of CD and CD-GU (combination of CD and GU) extract were studied. In addition, the effects of glycyrrhizin and liquiritin, active ingredients of Glycyrrhiza triterpenes and Glycyrrhiza flavones respectively, on the pharmacokinetics of daphnetin were also investigated. MATERIALS AND METHODS: Five groups of rats were orally administered with CD extract, CD-GU extract, pure daphnetin, co-administration of daphnetin and glycyrrhizin as well as co-administration of daphnetin and liquiritin at the same single dose of daphnetin (20 mg/kg). The rat plasma concentrations of daphnetin were determined by our developed UPLC-MS/MS method. The pharmacokinetics of daphnetin in above groups were investigated and compared. RESULTS: Comparing with oral administration of CD extract, AUC and Tmax of daphnetin significantly increased after giving CD-GU (p<0.05). In addition, in comparison to daphnetin alone, co-administration of daphnetin with liquiritin significantly increased the AUC and Cmax of daphnetin for ~1.5-fold, while co-administered with glycyrrhizin showed limited impact on the pharmacokinetics of daphnetin. CONCLUSIONS: In this study, it was found that liquiritin, one of the major components of GU, significantly enhanced the bioavailability of the main component daphnetin in CD. In addition, the bioavailability of daphnetin in the CD-GU prescription was also significantly higher than that in CD alone, which could be due to liquiritin. Such results explained the mechanism of the increased efficacy in treating RA with the combined use of CD and GU.

An integrated strategy based on UPLC-DAD-QTOF-MS for metabolism and pharmacokinetic studies of herbal medicines: Tibetan "Snow Lotus" herb (Saussurea laniceps), a case study.

ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea laniceps Hand.-Mazz. (SL) has long been used under the herbal name Tibetan "Snow Lotus" for the treatment of rheumatoid arthritis, stomachache and dysmenorrhea in Tibetan folk medicine. Since herbal medicine (HM) is a synergistical system with multiple components, both of the metabolism and pharmacokinetic studies of HM are interdependent. This study aimed to develop an integrated strategy based on the UPLC-DAD-QTOF-MS technique for metabolism and pharmacokinetic studies of HM. MATERIAL AND METHODS: SL was used here as a test herb to verify the feasibility of the proposed strategy. SL was administered to rats, then, the blood plasma, urine and feces were analyzed to determine the metabolic profiles. Using our strategy, umbelliferone and scopoletin were evaluated to be the key bioactive components. Their pharmacokinetic parameters were measured and biotransformation pathways were elucidated. RESULTS: After oral administration of SL to rats, 17 components in blood, 10 components in urine and 2 components in feces were identified and characterized using our UPLC-DAD-QTOF-MS method. Umbelliferone, scopoletin and their metabolites were found to be the major components involved in the metabolism process. Literature reports also suggest that umbelliferone and scopoletin are responsible for the therapeutic effects of SL, thus these two components were selected as the active markers for pharmacokinetic study. In the test of validity, the established method presented good linearity with R(2)>0.99. The relative standard deviation value was below 13.9% for precision, and recovery studies for accuracy were found to be within the range 91.8-112.5%. CONCLUSION: The present strategy offers, simultaneously, precision in quantitative analysis (metabolism study) and accuracy in quantitative analysis (pharmacokinetic study) with greater efficiency and less costs, which is therefore reliably used for integrated metabolism and pharmacokinetic studies of HM.

Novel S-Gal(®) analogs as (1)H MRI reporters for in vivo detection of ß-galactosidase.

The quantitative assessment of gene expression and related enzyme activity in vivo could be important for the characterization of gene altering diseases and therapy. The development of imaging techniques, based on specific reporter molecules may enable routine non-invasive assessment of enzyme activity and gene expression in vivo. We recently reported the use of commercially available S-Gal(®) as a ß-galactosidase reporter for (1)H MRI, and the synthesis of several S-Gal(®) analogs with enhanced response to ß-galactosidase activity. We have now compared these analogs in vitro and have identified the optimal analog, C3-GD, based on strong T1 and T2 response to enzyme presence (ΔR1 and ΔR2~1.8 times S-Gal(®)). Moreover, application is demonstrated in vivo in human breast tumor xenografts. MRI studies in MCF7-lacZ tumors implanted subcutaneously in athymic nude mice (n=6), showed significant reduction in T1 and T2 values (each~13%) 2h after intra-tumoral injection of C3-GD, whereas the MCF7 (wild type) tumors showed slight increase. Thus, C3-GD successfully detects ß-galactosidase activity in vivo and shows promise as a lacZ gene (1)H MR reporter molecule.

Evaluation of percutaneous absorption of esculetin: effect of chemical enhancers.

Percutaneous transdermal absorption of esculetin (6,7-dihydroxycoumarin), an oxidative damage inhibitor, was evaluated by means of in vitro permeation studies in which vertical Franz-type diffusion cells and pig ear skin were employed. To determine the absorption of esculetin, we validated a simple, accurate, precise, and rapid HPLC-UV method. Additionally, the effects of several percutaneous enhancers were studied. Pretreatment of porcine skin was performed with ethanol (control vehicle), decenoic acid, oleic acid, R-(+)-limonene, and laurocapram (Azone®) (5% in ethanol, w/w, respectively). Pretreatment of skin with oleic acid or laurocapram led to statistically significant differences in the transdermal flux of esculetin with respect to controls. Of the two enhancers, laurocapram showed the greatest capacity to enhance the flux of esculetin across pig skin.

Simultaneous determination of esculin and its metabolite esculetin in rat plasma by LC-ESI-MS/MS and its application in pharmacokinetic study.

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operated in the negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of esculin and its metabolite esculetin in rat plasma. After addition of internal standards scopoletin, the plasma sample was pretreated by solid-phase extraction (SPE), and separated on a reversed phase C(18) column with a mobile phase of 0.01% formic acid in water (solvent A) and methanol (solvent B) using isocratic elution (A:B=20:80, v/v). The detection of target compounds was done in multiple reaction monitoring (MRM) mode. The MRM detection was operated in the negative ESI mode using the transitions of m/z 339.1 ([M-H](-))→176.7 for esculetin, m/z 176.9 ([M-H](-))→133.0 and m/z 191.0 ([M-H](-))→175.9 for scopoletin. The standard curves, which ranged from 25 to 3200 ng/mL for esculin with the lowest limit of quantification (LLOQ) of 0.25 ng/mL and from 1.25 to 160 ng/mL for esculetin with the LLOQ of 1.25 ng/mL, were fitted to a 1/x weighted quadratic regression model. The method also afforded satisfactory results in terms of the sensitivity, specificity, precision (intra- and inter-day, RSD<8.73%), accuracy, recovery as well as the stability of the analyte under various conditions. The method was successfully applied to study the pharmacokinetics of esculin and its metabolite esculetin in rat plasma after oral administration of esculin at a dose of 100mg/kg.

[Effects of glycyrrhiza extract on pharmacokinetics property of daphnetin in rats].

OBJECTIVE: To research the influence of glycyrrhiza extract on the pharmacokinetics characteristic parameters of daphnetin, which was aimed to explore the rationality of concert application of drugs. METHOD: The rats received intragastric administration of daphnetin and glycyrrhiza extract containing the same daphnetin respectively. The blood concentration of daphnetin was assayed by LC-MS. The data was processed by program DAS2.1.1. RESULT: Glycyrrhiza extract can reduce the t(1/2), tmax and Ke of daphnetin, while increased the Ka and AUC(0-infinity). CONCLUSION: Glycyrrhiza extract promoted the oral absorption of daphnetin, slowed down the elimination and increased the biological availability.

[Analysis of metabolites of daphnetin in the intestinal wall of rats by liquid chromatography and quatrupole-time of flight mass spectrometry].

In this study, daphnetin and its major metabolites in the intestinal wall of rats were identified by liquid chromatography and quatrupole-time of flight mass spectrometry. Perfusion fluid of duodenum, jejunum, ileum and colon were collected separately for 2 hours from the rat intestine following perfusion with daphnetin. The metabolites of daphnetin in the perfusion fluid of different intestine segments were analyzed by the liquid chromatography and quatrupole-time of flight mass spectrometry. It is shown that the parent drug daphnetin and four metabolites were found in the perfusion fluid of duodenum, jejunum and ileum. However, no metabolites were found in the colon. Among the four metabolites, two daphnetin sulfates (m/z 257) were first discovered as the phase II metabolites of daphnetin in rats, which revealed a new way of daphnetin metabolism in rats.

Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of daphnetin.

Daphnetin has been developed as an oral medicine for treatment of coagulation disorders and rheumatoid arthritis in China, but its in vitro metabolism remains unknown. In the present study, the UDP-glucuronosyltransferase (UGT) conjugation pathways of daphnetin were characterized. Two metabolites, 7-O-monoglucuronide daphnetin (M-1) and 8-O-monoglucuronide daphnetin (M-2), were identified by liquid chromatography/mass spectrometry and NMR when daphnetin was incubated, respectively, with liver microsomes from human (HLM), rat (RLM), and minipig (PLM) and human intestinal microsomes (HIM) in the presence of UDP-glucuronic acid. Screening assays with 12 human recombinant UGTs demonstrated that the formations of M-1 and M-2 were almost exclusively catalyzed by UGT1A9 and UGT1A6, whereas M-1 was formed to a minor extent by UGT1A3, 1A4, 1A7, 1A8, and 1A10 at a high substrate concentration. Kinetics studies, chemical inhibition, and correlation analysis were used to demonstrate that human UGT1A9 and UGT1A6 were major isoforms involved in the daphnetin glucuronidations in HLM and HIM. By in vitro-in vivo extrapolation of the kinetic data measured in HLM, the hepatic clearance and the corresponding hepatic extraction ratio were estimated to be 19.3 ml/min/kg b.wt. and 0.93, respectively, suggesting that human clearance of daphnetin via the glucuronidation is extensive. Chemical inhibition of daphnetin glucuronidation in HLM, RLM, and PLM showed large species differences although the metabolites were formed similarly among the species. In conclusion, the UGT conjugation pathways of daphnetin were fully elucidated and its C-8 phenol group was more selectively catalyzed by UGTs than by the C-7 phenol.

Selection and effect of ointment bases for preparing collagenase inhibitor ointment using high-performance liquid chromatography and Franz cell apparatus.

After the dorsal subcutaneous administration of injectable collagen implant derived from bovine dermis (Zyderm; INAMED, Santa Barbara, CA) to mice, ointments that contain 3 types of collagenase inhibitors, Esculetin (6,7-dihydroxy-2H-chromen-2-one), ONO-4817 [(2S,4S)-N-hydroxy-5-ethoxymethyloxy-2-methyl-4-(4-phenoxybenzoyl) amino-pentanamide], and MMI270 (CGS27023A) {N-hydroxy-2(R)-[(4-methoxysulfonyl)-(3-picolyl)-amino]-3-methylbutanamide hydrochloride monohydrate} were applied daily on the dorsal region of mice (injection site), and intradermal Zyderm was extirpated after 30, 60, and 90 days to measure the level of hydroxyproline. Furthermore, dermal tissue was examined by Azan staining and immunostaining. A significant difference was observed in the level of hydroxyproline in the Esculetin and the ONO-4817 ointment groups compared with that in the control group after 30 days. A significant difference was also observed in the level of hydroxyproline in the Esculetin ointment group compared with that in the control group after 60 and 90 days. Histologically, 90 days after the application of the ointment, dense localization of type III collagen was observed around the injected Zyderm in the group applied Esculetin ointment compared with the control group. Therefore, it was indicated that Esculetin suppressed the degradation of collagen, and further facilitated the qualitative changes that increased neo-collagen, and that the collagen implant with hypodermic injection remained on behalf of ointments contained within the collagenase inhibitors that were applied on the skin surface.