The anticarcinogen activity of β-arbutin on MCF-7 cells: Stimulation of apoptosis through estrogen receptor-α signal pathway,inflammation and genotoxicity

Molecular and Cellular Biochemistry - Arbutin is one of the active ingredients employed in cosmetics as a skin whitening agent. In the present study, the possible effects of arbutin on breast...     

Synthesis of Karahanaenone Derivatives and Their Inhibition Properties toward Tyrosinase and Superoxide Scavenging Activity†

Fourteen amides condensing with aminophenols, anisidines, or aniline were synthesized from karahanaenone 1 as the starting material. The tyrosinase inhibitory activity and superoxide scavenging activity of these derivatives were examined in order to develop whitening agents for cosmetics. Of the compounds, N-p-hydroxyphenyl-1,3,3,6-trimethyl-5-cyclohepten-2-on-1-carboxamide 9, 2-hydroxy-N-o-hydroxyphenyl-3,3,6-trimethyl-5-cyclohepten-1-carboxamide 13, and 2-hydroxy-N-p-hydroxyphenyl-3,3,6-trimethyl-5-cyclohepten-1-carboxamide 15 showed strong tyrosinase inhibitory activity. 13 and 5 possessed a hydroxy group in the karahana skeleton and on the aromatic ring, respectively. These inhibitory rates were higher than that of arbutin that is used for commercial cosmetics (77.4%, 73.6%, and 72.3% against 63.0% for arbutin). Furthermore, 13 indicated 51.0% for superoxide scavenging activity.     

Simultaneous blood and brain sampling of cephalexin in the rat by microdialysis and microbore liquid chromatography: application to pharmacokinetics studies

To circumvent the need for laborious sample clean-up and multiple blood sampling, a system was developed consisting of on-line microdialysis coupled to microbore liquid chromatography and ultraviolet detection. The system was designed for the simultaneous and continuous monitoring of unbound blood and brain cephalexin in the rat following single bolus intravenous administrations (10 mg/kg, n=6). Microdialysis probes were inserted into the jugular vein and brain striatum, respectively, for blood and brain sampling. Chromatographic conditions consisted of a mobile phase of methanol–100 mM monosodium phosphoric acid (20:80, v/v, pH 5.0) pumped through a microbore reversed-phase column at a flow-rate of 0.05 ml/min. Detection wavelength was set at 260 nm. The method was validated for response linearity as well as intra- and inter-day variabilities. Rapid appearance of cephalexin in the striatal dialysate suggested good blood–brain barrier penetration. This study provided pharmacokinetics information for cephalexin as well as demonstrated the applicability of this continuous sampling method for pharmacokinetics studies.     

New trend in sample preparation: on-line microextraction in packed syringe for liquid and gas chromatography applications. I. Determination of local anaesthetics in human plasma samples using gas chromatography-mass spectrometry

A new technique for sample preparation on-line with LC and GC-MS assays was developed. Microextraction in a packed syringe (MEPS) is a new miniaturised, solid-phase extraction technique that can be connected on-line to GC or LC without any modifications. In MEPS approximately 1mg of the solid packing material is inserted into a syringe (100-250 microl) as a plug. Sample preparation takes place on the packed bed. The bed can be coated to provide selective and suitable sampling conditions. The new method is very promising. It is very easy to use, fully automated, of low cost and rapid in comparison with previously used methods. This paper presents the development and validation of a method for microextraction in packed syringe MEPS on-line with GC-MS. Local anaesthetics in plasma samples were used as model substances. The method was validated and the standard curves were evaluated by the means of quadratic regression and weighted by inverse of the concentration: 1/x for the calibration range 5-2000 nM. The applied polymer could be used more than 100 times before the syringe was discarded. The extraction recovery was between 60 and 90%. The results showed close correlation coefficients (R>0.99) for all analytes in the calibration range studied. The accuracy of MEPS-GC-MS was between 99 and 115% and the inter-day precision (n=3 days), expressed as the relative standard deviation (R.S.D.%), was 3-10%.     

New trends in sample preparation: on-line microextraction in packed syringe (MEPS) for LC and GC applications Part III: Determination and validation of local anaesthetics in human plasma samples using a cation-exchange sorbent, and MEPS-LC-MS-MS

The need for on-line sample preparation for high-throughput applications in bioanalysis has increased during the past decade. In this paper a robust and on-line sample preparation technique, micro extraction in packed syringe (MEPS) has been developed and validated. The method is a miniaturized, fully automated, solid-phase extraction (SPE) technique that can be connected on-line to GC or LC without any modification of the chromatographs. The performance of MEPS as sample preparation method is illustrated by the determination of local anaesthetics in human plasma samples on-line with high performance liquid chromatography (HPLC) and tandem mass spectrometry. The sampling sorbent was 1mg silica based benzenesulphonic acid cation exchanger that was inserted in a 250 microl syringe. Ropicavine and two of its metabolites (PPX and 3-OH-ropivacine), lidocaine and bupivacine were used as model substances. The accuracy values of quality control samples (QC) were between 95% and 109%, and precision (relative standard deviation, R.S.D.) had a maximum deviation of 9% for the analytes.     

Monitoring of ethanol during fermentation of a lignocellulose hydrolysate by on-line microdialysis sampling, column liquid chromatography, and an alcohol biosensor

During a 70-h fermentation of a lignocellulose hydrolysate, the ethanol produced was monitored on-line using a microdialysis probe as an in situ sampling device. The dialysate components were then separated in a column liquid chromatographic system and the ethanol was selectively detected by an amperometric alcohol biosensor. The result was compared with two off-line analysis methods: one chromatographic method with refractive index (RI) detection and one enzymatic method based on spectrophotometric detection. The two methods base on enzymes were shown to give lower values than the chromatographic method based on RI detection, which is discussed n terms of selectivity. The investigated on-line setup was found to be a flexible system for monitoring of fermentations, allowing a sampling frequency of at least 12 h(-1) and with a delay between sampling and detection of less than 5 min. (c) 1994 John Wiley & Sons, Inc.     

On-line derivatization for continuous and automatic monitoring of brain extracellular glutamate levels in anesthetized rats: a microdialysis study

Glutamate is an important excitatory amino acid in central nervous system. We developed a method for in vivo, continuous and automatic monitoring of brain extracellular glutamate, as well as other amino acids in anesthetized rat. This method involves the use of microdialysis perfusion technique and a high-performance liquid chromatography system equipped with a fluorescence detector. The microdialysate (perfused at a flow-rate of 1 μl/min) was on-line derivatized with o-phthaldehyde (perfused at 2 μl/min) through a mixing tee prior to the injection onto the HPLC column. The efficiency of this on-line derivatization was equivalent to that performed with an off-line manner. The effect of cerebral ischemia (2 h) and reperfusion (2 h) in brain cortex of anesthetized rats was monitored using this method. In addition to glutamate, extracellular concentrations of other amino acids, such as aspartate, glutamine, glycine, taurine and γ-aminobutyric acid, were also simultaneously monitored with this on-line method. Since monitoring of extracellular amino acids by microdialysis perfusion is intensively used in neuroscience investigations, this simple and convenient method would be useful in the future applications.     

Quantitation of nitric oxide-derived nitrite from activated macrophages using microdialysis sampling

An HPLC method for detecting nitrite in microdialysis samples obtained from activated RAW 264.7 macrophages in cell culture has been developed. Nitrite was quantified using a pre-column derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by HPLC-UV analysis of the azide product. For dialysates, the detection limit of nitrite was 750 nM and the quantitation limit was 2.5 microM. The microdialysis relative recovery of nitrite in the macrophage cell culture medium was determined to be 86+/-2% (n=3) at a flow rate of 0.7 microl/min. Nitrite produced from activated macrophages was measured immediately after lipopolysaccharide (LPS) stimulation using microdialysis sampling.     

Measurement and pharmacokinetic analysis of unbound ceftazidime in rat blood using microdialysis and microbore liquid chromatography

To evaluate the biodisposition of ceftazidime in rat blood, a rapid and simple microbore liquid chromatographic technique together with a microdialysis sampling technique were developed. This method involves an on-line design for blood dialysate directly injected into a microbore liquid chromatographic system. The chromatographic conditions consisted of a mobile phase of methanol–acetonitrile–100 mM monosodium phosphoric acid (pH 3.0) (10:10:80, v/v/v) pumped through a microbore reversed-phase column at a flow-rate of 0.05 ml/min. With the detection wavelength set at 254 nm, a good linear correlation was observed between the peak area and the ceftazidime concentration at 0.1 to 50 μg/ml (r=0.999). Microdialysis probes, being custom-made, were screened for acceptable in vivo recovery while chromatographic resolution and detection were validated for response linearity, as well as intra-day and inter-day variabilities. This method was then applied to the pharmacokinetic profiling of ceftazidime in blood following intravenous 50 mg/kg administration to rats. The pharmacokinetics was calculated from the corrected data for dialysate concentrations of ceftazidime versus time. This method has been used to study ceftazidime pharmacokinetics in rats and has proven to be rapid and reproducible.     

Measurement of hydroxyl radical in rat blood vessel by microbore liquid chromatography and electrochemical detection: an on-line microdialysis study

Salicylic acid (0.5 mM) is used as a trapping reagent of hydroxyl radical, and the formed 2,3- and 2,5-dihydroxybenzoic acids were collected via an on-line microdialysis device from the blood vessels. This study revealed the use of a sensitive liquid chromatographic system with electrochemical detection for the determination of 2,3- and 2,5-dihydroxybenzoic acids. Mobile phase consisted of 0.1 M monochloroacetic acid, 10 mM EDTA, 0.5 mM sodium octylsulfate, 20% acetonitrile and 5% tetrahydrofuran in 1 l (pH 3.0 adjusted with 1 M NaOH), and the flow-rate of 0.05 ml/min were found to be optimum. Isocratic separation of these adducts on a microbore column (reversed-phase C₁₈, 150×1 mm I.D., 5 μm) was achieved within 10 min. The optimal applied potential of dihydroxybenzoic acids was set at 750 mV based on a hydrodynamic study. This method has the detection limits of 1.3 pmol/ml (or 0.2 ng/ml) for 2,3- and 2,5-dihydroxybenzoic acids in Ringer solution (at signal-to-noise ratio=3).     

魔芋中神经酰胺类物质的HPLC-ELSD分析及其含量测定

建立高效液相色谱-蒸发光散射检测器分析神经酰胺的方法并进行了含量的测定.色谱柱:ZORBZX Eclipse XDB-C18(4.6mm×250mm,5μm),洗脱方法:梯度洗脱,柱温:35℃,流动相:甲醇/水,流速:1ml/min;检测器:蒸发光散射检测器,漂移管温度:40℃,氮气流速:1.5L/min.系统探讨了梯度洗脱的起始浓度、洗脱的时间和洗脱梯度的程序设置,最佳的梯度洗脱条件为5min内,甲醇浓度从60%线性增加为90%,从5min到25min,甲醇浓度线性增加为95%,在此条件下样品和标准品的分离色谱峰对称性较好.随后测定了各种样品中神经酰胺的含量,并进行了方法学验证,结果神经酰胺在0.2～2μg之间线性关系良好,最低检测限为0.01mg/ml,R2=0.9992;平均回收率为93.3%,RSD=1.65%(n=5).本法灵敏、方便、准确,重现性好,可用于魔芋神经酰胺类物质的分离及其含量的测定.     

Validation of a quantitative assay of arbutin using gas chromatography in Origanum majorana and Arctostaphylos uva‐ursi extracts

Introduction – Arbutin is a skin‐whitening agent that occurs naturally in the bark and leaves of various plants. It is commonly quantified in plant extracts and skin‐whitening products by HPLC. Objective – To develop an alternative gas chromatographic method for the separation and quantification of arbutin in Origanum majorana and Arctostaphylos uva‐ursi extracts. Methodology – N,O‐Bis(trimethylsilyl)acetamide and trimethylchlorosilane were used as silylation reagents, and the gas chromatographic separation of silylated extracts and standards was performed using a DB‐5 narrow bore column. GC‐MS was used for the compound identification, and the quantification was carried out by GC‐FID. The quantitative results were compared with those of HPLC analysis. Results – The developed method gave a good sensitivity with linearity in the range 0.33–500 mg/mL and recovery >98%, allowing the quantification of arbutin in O. majorana and A. uva‐ursi extracts. The relative standard deviations (RSD) relating to intra‐day and inter‐day precision were <0.002% and <4.8%, respectively. The GC results correlated well with those obtained by HPLC analysis. Conclusion – The analysis of marjoram and bearberry samples showed that the established GC method was rapid, selective, and demonstrated that arbutin could be screened alternatively by gas chromatography. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of lorazepam in plasma from children by high-performance liquid chromatography with UV detection

A simple, sensitive, selective, and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of lorazepam (LZP) in human plasma, using oxazepam (OZP) as internal standard. LZP and OZP were extracted from alkalinized (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with a mixture of n-hexane-dichloromethane (70:30%; v/v). Chromatographic separation was performed on a reversed-phase Synergi Max RP analytical column (150 mmx4.6 mm i.d.; 4 microm particle size), using an aqueous mobile phase (10 mM KH2PO4 buffer (pH 2.4)-acetonitrile; 65:35%, v/v) delivered at a flow-rate of 2.5 ml/min. Retention times for OZP and LZP were 10.2 and 11.9 min, respectively. Calibration curves were linear from 10 to 300 ng with correlation coefficients (r2) better than 0.99. The limits of detection (LOD) and quantification (LOQ) were 2.5 and 10 ng/ml, respectively, using 0.5 ml samples. The mean relative recoveries at 20 and 300 ng/ml were 84.1+/-5.5% (n=6) and 72.4+/-5.9% (n=7), respectively; for OZP at 200 ng the value was 68.2+/-6.8% (n=14). The intra-assay relative standard deviations (R.S.D.) at 20, 150 and 270 ng/ml of LZP were 7.8%, 9.8% (n=7 in all cases) and 6.6% (n=8), respectively. The inter-assay R.S.D. at the above concentrations were 15.9%, 7.7% and 8.4% (n=7 in all cases), respectively. Intra- and inter-assay accuracy data were within the acceptance interval of +/-20% of the nominal values. There was no interference from other commonly co-administered anticonvulsant, antimicrobial, antipyretic, and antimalarial drugs. The method has been successfully applied to a pharmacokinetic study of LZP in children with severe malaria and convulsions following administration of a single intravenous dose (0.1 mg/kg body weight) of LZP.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C₁₈ reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 μg/ml for plasma, 1.6 μg/g for muscle tissue and 0.5 μg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.     

Analysis of microsomal metabolic stability using high-flow-rate extraction coupled to capillary liquid chromatography-mass spectrometry

A method is described for on-line high-speed extraction of microsomal samples and analysis by capillary liquid chromatography-mass spectrometry (LC-MS) for the determination of metabolic stability in connection with the development of positron emission tomography (PET) tracers. The method allowed direct injections of large sample volumes at a fast extraction rate, providing a gain in both sensitivity and sample preparation time. The calibration curve of the test compound flumazenil (Ro 15-1788) was linear in the concentration range of 1-150 nM, with a correlation coefficient exceeding 0.999. The accuracy of the method ranged from 98 to 101%. A high precision was obtained, with mean intra-assay and inter-assay relative standard deviations of at most 1.4 and 1.5%, respectively, for quality control (QC) samples. The extraction efficiency was determined to be 99.4%, the total recovery 96% and the carryover to 相似文献   

Endoscopic ultrasound-guided tissue sampling by combined fine needle aspiration and trucut needle biopsy: a prospective study

BACKGROUND AND AIMS: Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has a diagnostic accuracy of 70-90%, depending on the site under evaluation. In order to improve EUS-guided tissue sampling a novel 19-gauge trucut-type needle has been designed to obtain core biopsies during EUS. We prospectively evaluated the safety and accuracy of EUS-FNA alone versus combined EUS-FNA and trucut needle biopsy (TNB) in patients referred to our Unit over a 3-year period. PATIENTS AND METHODS: A total of 159 patients underwent EUS-FNA alone (lesions<2 cm) or the combination of both sampling modalities (lesions>or=2 cm). The adequacy of sampling, sensitivity, specificity and overall accuracies of EUS-FNA or EUS-TNB alone and combined EUS-FNA/TNB were determined. RESULTS: Adequate samples were obtained by EUS-FNA, EUS-TNB and EUS-FNA/TNB in 91%, 88% and 97% of patients, respectively. From the pancreas (n=83), adequate samples were obtained by FNA in 94% and by TNB in 81%, compared with 87% and 92% from non-pancreatic sites (n=76), respectively. The combination of both techniques resulted in more adequate samples from non-pancreatic cases than EUS-FNA alone (P=0.044). The specificity was 100%. Overall accuracy for EUS-FNA alone was 77%, for EUS-TNB alone 73% and for EUS-FNA/TNB 91% (P=0.008). For pancreatic sampling, the accuracy of EUS-FNA alone was 77%, for EUS-TNB alone 56% and for EUS-FNA/TNB 83%. For non-pancreatic sampling, the accuracy for EUS-FNA alone was 78%, for EUS-TNB alone 83% and for EUS-FNA/TNB 95% (P=0.006). The complication rate was 0.6%. CONCLUSIONS: Combined EUS-FNA/TNB for lesions>or=2 cm improves adequacy of sampling and diagnostic accuracy compared with either technique alone and is safe.     

Identification of oxidation product of arbutin in mushroom tyrosinase assay system

In order to elucidate whitening mechanisms of arbutin (hydroquinone-O-beta-D-glucopyranoside), its effects on mushroom tyrosinase were analyzed by spectrophotometric, polarographic, and HPLC experiments. It was found that as soon as catalytic amounts of L-DOPA become available as a cofactor, arbutin acts as a monophenol substrate. A significant enzymatic product was identified as 3,4-dihydroxyphenyl-O-beta-D-glucopyranoside by NMR and MS experiments.     

BioMEMS device with integrated microdialysis probe and biosensor array

The fabrication of a microdevice for continuous sampling and on-line monitoring of glucose is described. The device comprised a microdialysis sampling system integrated on the flow through channel of a microfabricated enzyme sensor. The sensor was produced by thin film technology and was assembled to a printed circuit board (PCB) that provided the means for both electrical and fluidic connections. A polyacrilonitrile fibre, with a cut-off of 50 kDa, was used in the fabrication of the sampling probe. The performance of the device was evaluated in-vitro. High sampling efficiency of the microdialysis probe was achieved by appropriate selection of the perfusion fluid flow rate. Response times varying from 1.5 to 3.0 min were determined for flow rates ranging between 1 and 0.2 micro l/min. The linear response range was up to 30 mM glucose and interference from other electroactive substances was almost negligible. The device showed excellent stability under continuous operation for at least 5 days and sensitivity variation less than 3% over a period of 15 days.     

Sampling Rhodnius neglectus in Mauritia flexuosa palm trees: a field study in the Brazilian savanna

Two sampling methods (manual capture and live-baited adhesive traps) were compared for collecting the bug Rhodnius neglectus Lent (Hemiptera: Reduviidae: Triatominae) from palm trees, Mauritia flexuosa L. (Arecaceae), in the savanna of Brasília DF. R. neglectus was found in 19/50 (38%) of palm trees sampled. The detection rate was much higher by visual inspection and manual capture (18/50=36%) than by our trapping method (5/50=10%), although one tree was found to be positive by trapping but not by manual capture. Bugs collected manually were mostly (146/154=95%) found among the dead organic material in palm crowns. In combination, these sampling techniques are useful for quick detection of triatomine bug infestation in palm trees, especially in areas of high ecological value where the palms should not be cut and dissected, but arboreal Rhodnius are suspected to transmit enzootic Trypanosoma cruzi that might represent a risk of causing human cases of Chagas disease.     

Inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultraviolet-induced pigmentation

A pomegranate extract (PE) from the rind containing 90% ellagic acid was tested for its skin-whitening effect. PE showed inhibitory activity against mushroom tyrosinase in vitro, and the inhibition by the extract was comparable to that of arbutin, which is a known whitening agent. PE, when administered orally, also inhibited UV-induced skin pigmentation on the back of brownish guinea pigs. The intensity of the skin-whitening effect was similar between guinea pigs fed with PE and those fed with L-ascorbic acid. PE reduced the number of DOPA-positive melanocytes in the epidermis of UV-irradiated guinea pigs, but L-ascorbic acid did not. These results suggest that the skin-whitening effect of PE was probably due to inhibition of the proliferation of melanocytes and melanin synthesis by tyrosinase in melanocytes. PE, when taken orally, may be used as an effective whitening agent for the skin.