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ABSTRACT: A high-performance liquid chromatography-diode array detection/electrospray ionization mass spectrometry (HPLC-DAD/ESI-MS) method was applied to the characterization of ten iridoid glycosides in Gardenia jasminoides Ellis, a traditional Chinese medicine. During the process of structural elucidation, two groups of isomers including two epimers were structurally characterized and differentiated according to their distinctive fragmentation patterns which were closely related to their isomeric differentiations. Subsequently, the major compounds were purified by multi-dimensional chromatography and semi-preparative HPLC and the structure identification was confirmed with NMR techniques. The major fragmentation pathways of iridoid glycosides in Gardenia jasminoides Ellis obtained through the MS data were schemed systematically, which provided the best sensitivity and specificity for characterization of the iridoid glycosides especially the isomers so far. Based on the fragmentation patterns of iridoid glycosides concluded, seven major iridoid glycosides were characterized in rat plasma after intravenous administration of Gardenia jasminoides Ellis.
Rapid Communications in Mass Spectrometry 09/2010; 24(17):2520-8. · 2.79 Impact Factor
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ABSTRACT: Further studies of active coumarin components in Radix Angelicae Dahuricae (AE) are absolutely essential to provide data on pharmacology, toxicology and quality for innovative drug candidates. Thus, the preparation of active component standards and the administration of coumarin monomers should be carried out. The isolation of the low-level active components from complex Traditional Chinese Medicine (TCM) samples necessitates the development of rapid, simple and economical modern extraction, separation, identification and purification methods.
To develop an efficient strategy for the rapid extraction, separation, identification and purification of coumarins from AE.
First, active coumarins in AE were extracted with microwave-assisted extraction (MAE) after the extraction conditions were optimised. Second, gradient extraction methods with MAE were used to partially purify AE. Third, a high-performance liquid chromatography-diode array detection-electrospray ionisation tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) method was applied for the preliminary on-line identification and screening of the main coumarins in AE extract. Finally, a two-dimensional preparative high-performance liquid chromatography-diode array detection (2D-prep-HPLC-DAD) system was developed for further preparative separation of those target components.
Altogether 10 coumarins have been identified and five of them including xanthotoxol, osthenol, oxypeucedanin hydrate, byakangelicin and imperatorin were deemed as target components for the preparative isolation. All of the five isolated coumarins were at high purities of over 99% and the production rate was much higher than the traditional methods.
The present paper demonstrates that these consecutive approaches are very useful for to isolate chemical constituents from TCM.
Phytochemical Analysis 09/2010; 21(5):473-82. · 2.63 Impact Factor
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ABSTRACT: The influence of two different dietary patterns on maternal fatty acid (FA) intake on the composition of umbilical cord blood plasma phospholipids and transitional breast milk was investigated. A 7-day dietary record was completed in the last trimester of pregnancy by women living in an inland and a coastal area of south-eastern China. The FA composition in maternal diet was calculated using the 2002 Chinese food composition database. Cord blood and transitional breast milk samples were collected and their FA composition was analyzed by capillary gas-liquid chromatography. Mothers in the coastal area showed higher intake of long-chain polyunsaturated FA (LCPUFA) including docosahexaenoic acid (DHA, 22:6omega) and eicosapentaenoic acid (EPA,20:5omega3) but lower linoleic acid (LA, 18:2omega6) and alpha-linolenic acid (ALA, 18:3omega3) than the mothers in the inland area. The intake of arachidonic acid (AA, 20:4omega6) did not differ between the two areas. LA, ALA, AA and DHA in breast milk of day 5 reflected the maternal diet except that the EPA content in breast milk at day 5 was similar for the areas. LA, ALA and AA were lower and EPA higher in umbilical cord plasma phospholipids in infants from the costal compared to the inland area. There were significant differences in maternal intakes of FA confirming different dietary habits, which influenced the FA composition of cord plasma phospholipids and transitional breast milk. Since FA influence gene expression the found variation implies that the long-term follow-up of this cohort will be interesting.
Prostaglandins Leukotrienes and Essential Fatty Acids 09/2009; 81(5-6):325-30. · 3.37 Impact Factor
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ABSTRACT: A rapid HPLC method using a monolithic column with fluorescence detection has been developed for determination of telmisartan in human plasma. Sample preparation was done by protein precipitation with acetonitrile and naproxen was used as IS. The compounds were detected by fluorescence detection, using an excitation wavelength of 300 nm and emission wavelength of 385 nm. Calibration curves of telmisartan were linear in the range of 1-200 ng/mL. The assay was high throughput, sensitive and precise, and it was successfully applied to a bioequivalence study of two formulations of telmisartan.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2009; 877(29):3729-33. · 2.78 Impact Factor
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ABSTRACT: An ion-pair reverse-phase high performance liquid chromatographic method with UV-vis detection has been developed for the determination of total free iodine in rabbit plasma after vaginal administration of povidone-iodine (PVP-I). Sample preparation was done by protein precipitation with acetonitrile in 96-well format and aspirin was used as the internal standard. The 100 microL sodium thiosulfate solution (5 g L(-1)) was added to 100 microL plasma sample before protein precipitation, to convert the total free iodine in plasma to iodide (I(-)). Separation was performed on a C(18) column (200 x 4.6 mm i.d., 5 microm). The mobile phase consisting of a mixture of water phase (containing 10 mmol L(-1) 18-crown-6 ether, 5 mmol L(-1) octylamine and 5 mmol L(-1) sodium dihydrogen phosphate, pH adjusted to 6.0 with phosphoric acid) and acetonitrile in the ratio 70:30 (v/v) was delivered isocraticly at a flow rate of 1.0 mL min(-1). The method was sensitive with a lower limit of quantification of 0.005 microg mL(-1), with good linearity (r(2) > 0.9990) over the linear range of 0.005-2 microg mL(-1). All the validation data, such as linearity, accuracy and precision, were within the required limits. The method was successfully applied to study the pharmacokinetic of PVP-I in rabbits after vaginal administration.
Biomedical Chromatography 06/2009; 23(11):1151-9. · 1.97 Impact Factor
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ABSTRACT: The pressurized capillary electrochromatography (pCEC) was utilized for the separation and determination of coumarins in Fructus cnidii extracts from 12 different regions. After a thorough study of analytical parameters such as acetonitrile content of the mobile phase, the concentration and pH of the buffer, and the applied voltage, a methodology was proposed to separate and determine six coumarins of F. cnidii extracts in less than 15 min. The experiments were performed in an in-house packed column with a monolithic outlet frit under the optimal conditions: pH 4.0 ammonium acetate buffer at 10 mM containing 50% acetonitrile at -6kV applied voltage. The calibration curves were linear in the range of 10.0-100.0 microg/mL for bergapten, 20.0-200.0 microg/mL for imperatorin, 5.0-400.0 microg/mL for osthole, 10.0-100.0 microg/mL for 2'-acetylangelicin, 10.0-200.0 microg/mL for oroselone, and 10.0-200.0 microg/mL for O-acetylcolumbianetin. The correlation coefficients were between 0.9967 and 0.9995. With this pCEC system, fingerprints of F. cnidii extracts were preliminarily established to distinguish three types of coumarins by characteristic peaks, and the quality of various sources of raw materials was evaluated by determining the contents of six coumarins.
Journal of pharmaceutical and biomedical analysis 06/2009; 50(5):695-702. · 2.45 Impact Factor
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ABSTRACT: A two-dimensional column-switching system without sample loop trapping, where two columns were operated via a six-port switching valve, was employed in the isolation and purification of five isoflavonoids from Rhizoma Belamcandae: tectoridin, iridin, tectorigenin, irigenin and irisflorentin. The introduction of the six-port switching value, instead of a sample loop, assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension were not restricted. Two configurations were tested in this study. In the first mode, only one column was used in the second dimension and two 'heart-cutting' fractions were transported to the same second-dimensional column. In the second mode, two parallel columns were used in the second dimension and two fractions were transported to the two columns. Between the two configurations, the one with two second dimensional columns had double sample size, better resolution and one more purified compound. Both two-dimensional configurations consumed less solvent with even greater efficiency and shorter cycle time compared with conventional gradient methods. All of the isoflavonoids were isolated at high purities of greater than 95% with yields of above 82%.
Biomedical Chromatography 05/2009; 23(10):1064-72. · 1.97 Impact Factor
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ABSTRACT: Associated with many molecules, metastasis includes cell adhesion to extracellular matrix, migration towards specific direction and invasion into local vessel of distant organs. The purpose of the present study was to evaluate the role of ecto-5'-nucleotidase (eN, ecto-5-NT, CD73) generated extracellular adenosine in biologically malignant behaviors of human breast cancer cell lines.
Two human breast cancer cell lines, T-47D with lower expression of CD73 and MB-MDA-231 with higher expression of CD73, were used to investigate the functions of CD73. The effects of CD73 over-expression on invasion, migration and adhesion were observed in T-47D transfected with pcDNA-NT5E plasmid. The effects of specific CD73 inhibitor, alpha, ss-methylene ADP (APCP), were observed in MB-MDA-231 cells.
The results showed CD-73 overexpression increased invasion, migration and adhesion to ECM of the pcDNA-NT5E transfected T-47D cells compared to the saline and mock vector controls. The increased cell mobility of CD-73-overexpressed T-47D cells was blocked by APCP. Adenosine increased the mobility of wild type T-47D cells. APCP inhibited the mobility of the MB-MDA-231 cells.
Taken together, our results indicated that CD73 may facilitate the adhesion, migration and invasion of human breast cancer cells through its enzyme activity of generating adenosine. This study provided a possibly molecular mechanism of metastasis of breast carcinoma.
Journal of Cancer Research and Clinical Oncology 04/2008; 134(3):365-72. · 2.56 Impact Factor
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ABSTRACT: A two-dimensional column-switching system without sample loop trapping, where two columns were switched directly via a six-port two-position switching valve, was successfully applied for the first time to the isolation and purification of six iridoid glycosides including geniposide, gardenoside, shanzhiside, scandoside methyl ester, deacetyl-asperulosidic acid methyl ester and genipin-1-beta-D-gentiobioside from Gardenia jasminoides Ellis, a plant used in the traditional Chinese medicine. The introduction of the six-port switching valve instead of sample loop assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension could reach 20 ml. In this mode of operation, the sample size of the two-dimensional approach was more than 1.3 times that of conventional gradient methods with even less solvent consumption. And the simultaneous operations of the two dimensions allowed the cycle time to be less than 19 min, compared with that (90 min) in the gradient elution single-dimension mode of operation. All of the six isolated iridoid glycosides were isolated at high purities of over 99% with approximately 96% recoveries.
Journal of Chromatography B 11/2007; 858(1-2):296-301. · 2.89 Impact Factor
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ABSTRACT: An analytical method incorporating high-performance liquid chromatography (HPLC) with MS and UV-detection was developed for the qualitative and quantitative determination of alkaloids in Corydalis yanhusuo. Ten alkaloids, including seven tertiary alkaloids and three quaternary alkaloids, were identified by comparing their retention times, UV and MS spectra with those of authentic compounds. Furthermore, the collision-induced dissociations of the [M+H](+) and [M](+) ions were studied to clarify the MS behavior of the different types of alkaloids. In positive ion electrospray ionization tandem mass spectrometry (ESI-MS) all the tertiary alkaloids yielded prominent [M+H](+) ions and quaternary alkaloids yielded prominent [M](+) ions in the first order mass spectra. Fragments involving losses of H, CH(3), CO, H(2)O and OCH(3) were observed in the MS/MS spectra. In addition, quantification of the 10 alkaloids in Corydalis yanhusuo from methanol and ethyl acetate extract of different origins were performed by this method, which provides a new tool for the assessment of quality of Corydalis yanhusuo preparations. The method provides the best sensitivity and specificity for characterization and quantitative determination of the alkaloids in Corydalis yanhusuo so far.
Journal of Pharmaceutical and Biomedical Analysis 11/2007; 45(2):219-26. · 2.97 Impact Factor
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ABSTRACT: The dried flower buds of Magnolia biondii Pamp are one of the most widely used medicinal plants officially listed in the Chinese Pharmacopoeia. A 2-D column-switching system without sample loop trapping, where two columns were switched directly via a six-port two-position switching valve, was successfully applied for the first time to the isolation and purification of five lignans including pinoresinol dimethyl ether, magnolin, epi-magnolin A, fargesin, and demethoxyaschantin from M. biondii Pamp after microwave-assisted extraction. The introduction of the six-port switching valve instead of sample loop assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension could reach 12 mL. In this mode of operation, the solvent consumption of the 2-D approach was less than 30% that of conventional gradient methods with even larger sample size. The simultaneous operations of the two dimensions allowed the cycle time to be less than 16 min, compared to 90 min in the gradient elution single-dimension mode of operation. All of the five lignans were isolated at high purities of over 99% with approximately 95% recoveries.
Journal of Separation Science 11/2007; 30(15):2370-81. · 2.73 Impact Factor
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ABSTRACT: An efficient on-line purity monitoring strategy based on on-line coupling of high-speed counter-current chromatography (HSCCC) with high-performance liquid chromatography-diode array detection (HPLC-DAD) was successfully applied for the first time to the isolation and purification of 5-hydroxymethyl-furancarboxaldehyde (5-HMF), mangiferin and neomangiferin from the Chinese medicinal plant Anemarrhena asphodeloides Bunge, a plant used in the traditional Chinese medicine. The introduction of on-line purity monitoring in HSCCC has greatly improved the efficiency of this technique by overcoming the drawbacks of post-purification sample handling in HSCCC isolation. The effluent from the outlet of HSCCC was split into two parts, and one was collected, while the other was introduced directly through a switch valve into a HPLC-DAD system for purity monitoring. Using this method the desired fractions with high purities could be collected. From 600 mg partially purified extract, 165.6 mg neomangiferin and 292.8 mg mangiferin with purities of 98.9 and 99.5%, respectively, were obtained with a two-phase solvent system composed of n-butanol-water (1:1, v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.2 ml min(-1) after 210 min. A 17.1mg 5-HMF with purity of 96.6% was also isolated for the first time.
Journal of Pharmaceutical and Biomedical Analysis 06/2007; 44(1):96-100. · 2.97 Impact Factor
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ABSTRACT: Ecto-5'-nucleotidase (CD73) was overexpressed in malignancies of epithelial origin and was involved in a variety of cellular processes such as cytoprotection and anti-inflammation. In the present study, human mammary T-47D cells were transfected with pcDNA-NT5E to establish a CD73 overexpressed cell model. Short small interfering RNA (siRNA) was used to silence the epidermal growth factor receptor (EGFR) gene. Real-time PCR, RT-PCR and western-blot were used to study CD73 and EGFR expression. Surface CD73 activity was assessed by quantifying the conversion of etheno-AMP to ethenoadenosine via HPLC. Interleukin (IL)-8 mRNA and protein expression were analyzed by RT-PCR and ELISA. Cell motility, invasiveness and adhesion to extracellular matrix (ECM) were measured by in vitro invasion and adhesion assay before and after transfection. We demonstrated that abilities of migration, invasions and adhesion to ECM in pcDNA-NT5E transfected T-47D cells increased significantly, which can be blocked by CD73 inhibitor alpha, beta-methylene ADP(APCP). Addition of adenosine reversed the effects of APCP. The mRNA and protein expression of EGFR and IL-8 increased in pcDNA-NT5E transfected T-47D cells. EGFR siRNA down-regulated EGFR expression dramatically, leading to migration and invasion activities inhibition in pcDNA-NT5E transfected T-47D cells. Our results suggest that up-regulated adenosine production, EGFR and IL-8 expression due to overexpressed CD73 may involved in CD73-promoted breast cancer metastasis.
Cancer biology & therapy 04/2007; 6(3):426-31. · 2.64 Impact Factor
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ABSTRACT: Following preparative isolation and purification by high-speed counter-current chromatography (HSCCC), the collected fractions were generally analyzed by high-performance liquid chromatography (HPLC) to determine the relative purities of each fraction. Our paper reports for the first time a preparative isolation-purity detection hyphenated system: online coupling of HSCCC with high-performance liquid chromatography-diode array detection (HSCCC-HPLC-DAD). The introduction of online purity analysis in HSCCC has dramatically improved the efficiency of this technique by overcoming the drawbacks of post analysis in HSCCC isolation. The effluent from the outlet of HSCCC was splitted into two parts: one was collected, while the other was introduced directly into an HPLC-DAD system for purity analysis through a switch valve. Therefore, the purities of the obtained fractions from HSCCC were monitored, and fractions with high purities were collected. This strategy has been successfully demonstrated with the preparative isolation and purification of hyperoside from Hypericum perforatum (St. Jone's Wort); a model of TBE-300A HSCCC was used to isolate and separate hyperoside from H. perforatum with a two-phase solvent system composed of ethyl acetate-ethanol-water at the volume ratio of 5:1:5 (v/v) using online detection technique. The isolation was done in less than 3.5 h, and a total of 83.0-mg hyperoside at over 99.0% purity was yielded from 300 mg of the partially purified extract. This new strategy possesses general utility in the preparation of bioactive compounds from traditional Chinese medicine (TCM).
Journal of Chromatography 06/2006; 1116(1-2):97-101. · 4.53 Impact Factor
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ABSTRACT: High-speed counter-current chromatography (HSCCC) was applied to the isolation and purification of geniposide from Gardenia jasminoides Ellis. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v). According to the above solvent system, preparative HSCCC was successfully performed with the optimal solvent system composed of ethyl acetate-n-butanol-water (2:1.5:3, v/v/v) yielding 389 mg of geniposide at over 98% purity from 1g of the partially purified extract with 38.9% recovery in a one-step separation.
Journal of Chromatography 01/2006; 1100(1):76-80. · 4.53 Impact Factor
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ABSTRACT: Cerebral ischemia and post-ischemic reperfusion commonly result in significant brain damage. Brain microvessel endothelial cells, the key target cells and regulating sites, can secrete adenosine which plays an important neuroprotective role in the ischemic brain. A primary determinant of localized production of adenosine at tissue interfaces is ecto-5'-nucleotidase (CD73). In our experiments, we used bEnd.3 cells, immortalized mouse brain microvessel endothelial cell lines, as the target cells to study the effect of hypoxia and posthypoxic reoxygenation on CD73 in brain microvessel endothelial cells. CD73 activity in bEnd.3 cells exposed to hypoxia significantly increased in time-dependent way. The upregulation of CD73 mRNA and protein expression induced by hypoxia in bEnd.3 cells were detected by RT-PCR and Western blot. However, for reoxygenation studies, CD73 activity, mRNA and protein expression decreased at the initial stages, but increased at prolonged reoxygenation. Our results suggest that hypoxia can induce upregulation of CD73 expression in brain microvessel endothelial cells, which can be reversed by reoxygenation of short duration. But CD73 expression increased gradually with the duration of reoxygenation. Then, we infer that CD73 in brain microvessel endothelial cells plays a very important role through forming adenosine during brain ischemia and reperfusion.
Microvascular Research 72(1-2):48-53. · 2.83 Impact Factor
