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Alismatis rhizoma (AR), the dried rhizome of Alisma orientale (Sam.) Juzep, is a well-known, traditional medicine that is used for the various biological activities including as a diuretic, to lower cholesterol and as an anti-inflammatory agent. The present study was carried out to investigate the potential toxicity of the Alismatis rhizoma aqueous extract (ARAE) following 90-day repeated oral administration to Sprague-Dawley rats. ARAE was administered orally to male and female rats for 90 days at 0 (control), 500, 1,000 and 2,000 mg/kg/day (n = 10 for male and female rats for each dose). Additional recovery groups from the control group and high dose group were observed for a 28-day recovery period. Chromatograms of ARAE detected main compounds with four peaks. Treatment-related effects including an increase in the red blood cells, hemoglobin, hematocrit, albumin, total protein, and urine volume were observed in males of the 2,000 mg/kg/day group (p < 0.05). However, the diuretic effect of ARAE was considered, a major cause of hematological and serum biochemical changes. The oral no-observed-adverse-effect level (NOAEL) of the ARAE was > 2,000 mg/kg/day in both genders, and no target organs were identified.

Background: Samsoeum (SSE), a Korean medicine, has been used to treat upper respiratory infection including residual coughs after catching a cold, and colds in patients with gastrointestinal disorder. In this study, we investigated the inhibitory effect of SSE against lipopolysaccharide (LPS)-induced bronchitis and characterized its optimal dosing range based on the improvement of SSE concentrations. Materials and methods: Male Sprague Dawley rats were intra-nasally administered LPS on day 0, 3 and 6. 2 g kg−1 dose of SSE for rat was determined by the human equivalent dose formula and orally administered once a day from day 3 to day 6. To clarify the optimal administration dose of SSE, various doses including 0.5 (1/4 fold), 1 (1/2 fold), 6 (3 fold), 12 (6 fold), 24 (12 fold) and 36 g kg−1 (18 fold) were also orally administered. In addition, the molecular mechanism of SSE in mucin hyperproduction was investigated in LPS-sensitized A549 cells. Results: Oral administration of SSE ameliorated alveolar wall thickening and inflammatory cell infiltration of lung tissues in LPS-induced bronchitis at doses of 1/4 fold, 1/2 fold and 1 fold. The total cell and neutrophil numbers in bronchoalveolar lavage fluid (BALF) were reduced in the SSE-treated groups compared with the LPS group. In addition, 0.5, 1 and 2 g kg−1 of SSE suppressed LPS-induced mucin glycoprotein 5AC (MUC5AC) production in BALF. Furthermore, SSE treatment significantly inhibited the pro-inflammatory cytokines, resulting in the decrease of MUC5AC production by the JAK1/STAT6 signaling pathway. Conclusions: 1, 2 and 6 g kg−1 of SSE ameliorated chronic bronchitis by inhibiting LPS-induced neutrophil infiltration and MUC5AC release in BALF. These findings suggested that SSE with 0.5–3-fold of general daily intake dose would be a therapeutic agent for chronic bronchitis.

The purpose of this study was to develop a method for simultaneous analysis of schizandrin, ephedrine, paeoniflorin, and cinnamic acid as constituents of Socheongryong-tang tablet in human plasma using UPLC-MS/MS. These four components were separated using water containing 0.01% formic acid and methanol as a mobile phase by gradient elution at a flow rate of 0.3 mL/min with a HALO-C18 column (2.1 mm × 100 mm, 2.7 μm particle size). Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique operated in multiple reaction monitoring mode. Mass transitions were m/z 432.9 → 384.1 for schizandrin, 165.8 → 148.1 for ephedrine, 525.0 → 449.2 for paeoniflorin, 146.8 → 102.9 for cinnamic acid, and 340.0 → 324.0 for papaverine as internal standard. Liquid-liquid extraction and protein precipitation with ethyl acetate-methanol (1:2, v/v) were used to obtain these four components. Chromatograms showed high resolution, sensitivity, and selectivity without interference by plasma constituents. Calibration curves of schizandrin, ephedrine, paeoniflorin, and cinnamic acid in human plasma ranged from 0.02 to 8 ng/mL, 0.5 to 200 ng/mL, 0.2 to 80 ng/mL, and 1 to 400 ng/mL, respectively. Calibration curves of each analyte displayed excellent linearity, with correlation coefficients > 0.99. For all four components, both intra- and inter-day precisions (CV%) were

Column chromatographic separation of the MeOH extract from the roots of Dicentra spectabilis yielded fourteen compounds, menisdaurin (1), menisdaurilide (2), trans-N-pcoumaroyltyramine (3), trans-N-p-feruloyltyramine (4), 4-Oferuloylquinicacid (5), chlorogenic acid (6), 3-O-feruloylquinicacid (7), ferulic acid (8), protopine (9), Kaempferol 3,7-di-O-β-Dglucopyranoside (10), kaempferol 3-O-β-D-glucopyranosyl-7-O- α-L-rhamnopyranoside (11), α-rhamnoisorobin (12), astragalin (13), and nicotiflorin (14). Their structures were determined on the basis of NMR spectroscopic data. Among them, compound 1, 3- 8, and 10-14 isolated from this plant were reported for the first time. The isolated compounds (1-14) were tested for nitric oxide (NO) inhibitory activity on lipopolysaccharide-stimulated RAW 264.7 cells. Compound 3, 4 and 12 significantly inhibited NO production. Moreover, Compound 3 suppressed pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in a dose- dependent manner. These data suggest that compound 3 possess anti-inflammatory activity and might be useful natural materials for development of anti-inflammatory agent.

So-Cheong-Ryong-Tang, which is a standardized Korean medicine of the National Health Insurance, is a traditional prescription for the treatment of allergic rhinitis, bronchitis, and bronchial asthma. Simultaneous analysis and development of SCRT is essential for its stability, efficacy, and risk management. In this study, a simple, reliable, and accurate method using ultrahigh-performance liquid chromatography (UPLC) fingerprinting with a diode array detector (DAD) was developed for the simultaneous analysis. The chromatographic separation of the analytes was performed by an ACQUITY UPLC BEH C18 column (1.7 μM, 2.1 × 100 mm, Waters) with a mobile phase of water containing 0.01% (v/v) phosphoric acid and acetonitrile containing 0.01% (v/v) phosphoric acid. The flow rate and detection wavelength were set at 0.4 mL/min and 215, 230, 254, and 280 nm. All calibration curves of the thirteen components showed good linearity (R2 > 0.999). The limit of detection and limit of quantification ranged 0.001–0.360 and 0.004–1.200 µg/mL, respectively. The relative standard deviation (RSD) of intra- and interday was less than 2.60%, and the recoveries were within the range 76.08–103.79% with an RSD value of 0.03–1.50%. The results showed that the developed method was simple, reliable, accurate, sensitive, and precise for the quantification of bioactive components of SCRT.

The aim of this study was to develop an analytical method to simultaneously analyze schizandrin, schizandrol B, and gomisin N lignans in human plasma using ultra high performance liquid chromatography with tandem mass spectrometry. The three lignans were separated using a mobile phase of water and acetonitrile containing 0.02% acetic acid equipped with a Kinetex C18 column (2.1 mm × 50 mm, 1.7 μm). This analysis was achieved by multiple reaction monitoring mode in an electrospray interface. The mass transitions were m/z 433.1→384.0 for schizandrin, 398.8→367.8 for schizandrol B, and 400.6→299.8 for gomisin N. Liquid-liquid extraction with methyl tert-butyl ether was used to obtain the three lignans. The chromatograms showed high resolution, sensitivity, and selectivity with no interference with plasma constituents. The calibration curves for the three lignans in human plasma were 0.05-50 ng/mL and displayed excellent linearity with correlation coefficients greater than 0.99. Precision for all three lignans was within 11.23%. The accuracy was 88.3-99.0% for schizandrin, 90.6-103.4% for schizandrol B, and 90.2-103.5% for gomisin N. The developed simultaneous analytical method satisfied the criteria of international guidance and could be successfully applied to the pharmacokinetic study of three lignans after oral administration of Schisandrae Fructus extract powder to humans.

An accurate, precise, selective, and sensitive liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of imperatorin (IMP) and its metabolite, xanthotoxol (XAN), in rat plasma and urine samples. The analytes, along with psoralen as an internal standard, were determined by multiple reaction monitoring (MRM) operated in the positive electrospray ionization (ESI) mode. Chromatographic separation was performed on an Acquity UPLC BEH C18 column (50mm×2.1mm, 1.7μm) with a mobile phase consisting of 0.1% formic acid solution and 0.1% formic acid in methanol at a flow rate of 0.3mL/min. The run time was 6min per sample and the injection volume was 5μL. The method had a lower limit of quantification (LLOQ) of 0.25ng/mL for IMP in plasma and urine, and 1ng/mL for XAN in urine. The linear calibration curves were fitted over the range of 0.25-1000ng/mL for IMP in plasma, 0.25-1000ng/mL for IMP in urine, and 1-1000ng/mL for XAN in urine, with correlation coefficients greater than 0.995. The inter- and intra-day accuracies (relative error, RE%) were between -8.5% and 3.5%, and the precisions (relative standard deviation, RSD%) were less than 10.0% for all quality control samples (QCs). The analytes were extracted from rat plasma and urine samples using a liquid-liquid extraction method with the extraction recovery in the range of 60.3-79.1%. A good stability of the analytes was observed in all the analysis procedures. The method was successfully validated and applied to determine the pharmacokinetics of IMP in rat plasma and, for the first time, the metabolite kinetics of IMP to XAN in rat urine after IMP administration.

Paeoniae Radix, a traditional herbal medicine, has been used in antispasmodic action, alleviating pain, and activating circulation. The aim of this study was to evaluate the pharmacokinetics (PKs) of paeoniflorin in healthy Korean subjects and develop an analytical method for the quantification of paeoniflorin in human plasma. This was an open-label, randomized, and single-dose study in 10 healthy Korean subjects who received a Paeoniae Radix extract powder. The plasma concentration of paeoniflorin up to 12 h was determined using a validated ultra-performance liquid chromatography tandem mass spectrometric method. The PK parameters such as AUC0−∞, CL/F, Vd/F, Cmax, Tmax and t1/2 were calculated using WinNonlin® software (version 6.4, Pharsight®, a Certara™ Company). The best PK model of paeoniflorin in humans was the one-compartment model. The mean parameters were 2625.71 L/h, 10,150.55 L, 6.97 ng/mL, and 1.64 h for the oral clearance (CL/F), the volume of distribution (Vd/F), the maximum plasma concentration (Cmax), and the time to reach Cmax(Tmax), respectively. The elimination half-life (t1/2) was 2.68 h. The validated method was successfully applied to the PK study of paeoniflorin in humans. The lower limit of quantification for paeoniflorin in human plasma was 0.2 ng/mL. This study was the first to evaluate the PKs of paeoniflorin after the usual oral dose of Paeoniae Radix extract powder (3.4 g containing 108.18 mg paeoniflorin) in Korean subjects.

Coumarins in Cham-dang-gwi, the dried root of Angelica gigas Nakai (AGN), possess pharmacological effects on anemia, pain, infection, and articular rheumatism. The AGN root containes decursin (D), decursinol angelate (DA), nodakenin, and decursinol (DOH), a major metabolite of D and DA. The aim of this study was to develop a simultaneous determination method for these four coumarins in human plasma using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Chromatographic separation was performed on dual columns (Kinetex® C18 column and Capcell core C18 column) with mobile phase consisting of water and acetonitrile at a flow rate of 0.3 mL/min using gradient elution. Multiple reaction monitoring was operated in positive ion mode with precursors to product ion transition values of m/z 328.9→228.8, 328.9→228.9, 409.4→248.8, and 246.8→212.9 to measure D, DA, nodakenin, and DOH, respectively. Linear calibration curves were fitted over concentration range of 0.05–50 ng/mL for these four components, with correlation coefficient greater than 0.995. Inter- and intra-day accuracies were between 90.60% and 108.24%. These precisions were within 11.19% for all components. The established method was then applied to a pharmacokinetic study for the four coumarins after usual dosing in Korean subjects.

A rapid, selective and sensitive ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry method about the simultaneous determination of puerarin and its major active metabolite, daidzein, in human plasma was developed and validated in order to investigate the pharmacokinetics (PKs) of Gegen after the usual oral dose administration to human. Chromatography was carried out on a Kinetex C18 column (2.1mm×50mm, 1.7μm) using 0.05% acetic acid in water and 0.05% acetic acid in methanol as mobile phase with a gradient elution. Liquid-liquid extraction with ethyl acetate in acidic condition could remove the interference and minimize the matrix effect of human plasma. The lower limit of quantification in human plasma was 0.2ng/mL for both of compounds, puerarin and daidzein. The calibration curves for puerarin and daidzein in human plasma were linear over all the concentration range of 0.2-100ng/mL with correlation coefficients greater than 0.998. This assay procedure was successfully applied to the PKs of puerarin and daidzein, after the usual oral dose of Gegen extract powder (2.56g, containing 9.984mg puerarin) in human subjects.

Baicalein, a flavonoid and aglycon hydrolyzed from baicalin, has anticancer properties in several human carcinomas, but its molecular mechanisms of action remain unclear. Here, we show that baicalein leads to human cancer cell death by inducing autophagy rather than apoptosis, because cell death induced by baicalein was completely reversed by suppressing the expression levels of key molecules in autophagy such as Beclin 1, vacuolar protein sorting 34 (Vps34), autophagy-related (Atg)5 and Atg7, but not by pan-caspase inhibitor. Our data revealed that baicalein significantly increased the number of green fluorescence protein-cytosol-associated protein light chain 3 (GFP-LC3)-containing puncta and LC3B-II expression levels, which were further enhanced by chloroquine treatment. Furthermore, a luciferase-based reporter assay showed that the ratio of RLuc-LC3wt/RLuc-LC3G120A was greatly reduced. The data suggested that baicalein induced not only autophagosome formation, but also autophagic flux. Experiments using short interfering RNAs and pharmacological inhibitors revealed that Beclin 1, Vps34, Atg5, Atg7 and UNC-51 (Caenorhabditis elegans)-like kinase 1 (ULK1) play pivotal roles in mediating baicalein-induced autophagy. Moreover, baicalein activated AMP-activated protein kinase (AMPK)α, leading to ULK1 activation through phosphorylation at Ser555, whereas both protein and mRNA levels of mammalian target of rapamycin (mTOR) and Raptor, upstream inhibitors of ULK1 and autophagy, were markedly downregulated by baicalein. Our data suggest that the anticancer effects of baicalein are mainly due to autophagic cell death through activation of the AMPK/ULK1 pathway and inhibition of mTOR/Raptor complex 1 expression. These results provide new mechanistic insights into the anticancer functions of autophagy inducers, such as baicalein, which may be used as potential therapeutics for cancer treatment.

This experiment was performed to evaluate the usefulness of an experimental fibrosis model by bile duct ligation as a pharmacokinetic model of a disease state. First, experimental liver fibrosis was produced by bile duct ligation. At 4 weeks postoperation, a fibrotic condition was characterized by measurement of the aminoterminal procollagen type III peptide (PIIINP) level in serum, total collagen content in liver and light microscopic histology. Four weeks after bile duct ligation there was an increase in total collagen content of the liver to 430% of the initial values, accompanied by an increase of serum-PIIINP (385%). Secondly, we examined the pharmacokinetics of theophylline in the fibrotic rat induced by bile duct ligation. An i.v. dose of 8 mg of theophylline per kg of body weight was administered, and the levels of theophylline in serum were assayed by high performance liquid chromatography. The area under the serum concentration-time curve of theophylline was increased significantly in fibrotic rats compared with that of the control, and the total clearance of drug in fibrotic rats was low, averaging 22.6 mg/kg/h vs. 36.1 and 60.9 ml/kg/h in the control and the normal rat, respectively. However, the value of distribution during the beta-phase was not significantly affected by experimental liver fibrosis.