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ABSTRACT: Liquiritigenin (7,4'-dihydroxyflavone), the primary active component of a traditional Chinese medicine Glycyrrhizae radix, has a wide range of pharmacological activities. Six oxidative metabolites of liquiritigenin (7,3',4'-trihydroxyflavone, a hydroxyl quinine metabolite, two A-ring dihydroxymetabolites, 7,4'-dihydroxyflavone, and 7-hydroxychromone) have been detected in rat liver microsomes (RLMs), and one CYP3A4-catalyzed metabolite (7,4'-dihydroxyflavone) has been identified in human liver microsomes (HLMs) recently. In this study, a novel mono-hydroxylated metabolite was detected in reaction catalyzed by HLMs, and was identified as 4',5,7-trihydroxyflavanone by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound. Significant difference in CL(int) (9-fold) was found between these two oxidative pathways of liquiritigenin, and C5-hydroxylation pathway was identified as the major oxidative metabolism of liquiritigenin. The study with chemical selective inhibitor, cDNA-expressed human CYPs, correlation assay, and kinetic study demonstrated that CYP1A2 was the specific isozyme responsible for the C5-hydroxylation metabolism of liquiritigenin in HLMs.
Xenobiotica 01/2011; 41(5):349-57. · 1.79 Impact Factor
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ABSTRACT: The distribution and level of yew constituents vary with species and tissues. In this study, a rapid and valid method incorporating ultra-fast liquid chromatography (UFLC) with MS and UV detection was developed for simultaneous determination of paclitaxel and its six semisynthesis precursors in needles and hair roots from various Taxus species. All target analytes could be identified by comparing their retention times as well as UV and MS spectra with authentic standards, while seven valuable taxanes in botanical samples can be rapidly determined by UFLC-DAD with excellent sensitivity. Analysis of more than one hundred yew samples from nine species showed significant variations in distribution and content of seven evaluated taxanes. Thus, different developmental schemes should be used for better utilization of various yew resources.
Planta Medica 10/2010; 76(15):1773-7. · 2.15 Impact Factor
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ABSTRACT: The traditional Chinese medicine formula Fuling Decoction (FD) has been clinically used for eczema treatment, but the unclear chemical distribution and the lack of quality control have strongly restricted its application. In this study, an analytical method incorporating ultra-fast liquid chromatography (UFLC) with MS and UV detection was developed for rapid profiling of the chemical constitutes from FD. Fourteen constitutes were identified by UFLC-ESI-MS, while four major components including genipingentiobioside, geniposide, paeoniflorin and liquiritin were quantified simultaneously by UFLC-DAD. The UFLC-based method was fully validated and can be applied to screening and determination of principal components in commercially FD prescriptions.
Fitoterapia 03/2010; 81(6):662-7. · 1.85 Impact Factor
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ABSTRACT: A simple and sensitive method for determination of the O-demethylation activity of rat, dog, minipig, and human liver micrsomes toward paeonol using ultra-performance liquid chromatography with mass detection (UPLC-MS) has been developed. The method uses chemically synthesized O-demethylated metabolite of paeonol (2,4-dihydroxyacetophenone, DHA) as a standard for method validation. Validation was done with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C(18) column (50 mm x2.1 mm i.d., 1.7 microm), with phase of acetonitrile-water (ratio 30:70). Selective ion reaction (SIR) monitor was specific for paeonol, DHA and I.S. The method was specific since there were no interference peaks from the reaction matrix. The calibration curve for DHA was linear from 0.5-100 microM with r(2)=0.9999. The newly developed method has good precision and accuracy. The method was successfully used to determine the kinetics of DHA activities toward paeonol in liver microsomes from different species. Dog liver microsomes (DLMs) were the most active in paeonol O-demethylation (709.7 pmol/min/mg protein) followed by rat liver microsomes (RLMs) (579.6 pmol/min/mg protein), HLMs (569.3 pmol/min/mg protein), and then minipig liver microsomes (PLMs) (417.3 pmol/min/mg protein). The developed method was appropriated for rapid screening paeonol O-demethylation activity in liver microsomes from different species.
Talanta 11/2009; 79(5):1433-40. · 3.79 Impact Factor
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ABSTRACT: The acceptor specificity and transfer potential of a beta-D-glycosidase (G I), which had been purified from the China white jade snail, were further investigated by using various sugars as acceptors. G I had broad monosaccharide acceptor specificity for its transglycosylation activity. More specifically, it efficiently catalyzed the transfer of the beta-D-fucosyl, beta-D-glucosyl or beta-D-galactosyl moiety from the corresponding p-nitrophenyl-beta-D-glycopyranosides to various monosaccharides. The transfucosylation efficiency of G I was studied by using p-nitrophenyl-beta-D-fucopyranoside (pNPFuc) as the donor and glucose and xylose as the acceptors. The yields under conditions of non-initial velocity were 88% for glucose and 93% for xylose. The transfer product with glucose as the acceptor was isolated and identified as beta-fucosyl-1,6-glucose by an NMR analysis. The data from these analyses indicate that G I had broad acceptor specificity and high efficiency for transglycosylation. These uncommon properties of G I could make it a valuable biocatalyst for the synthesis of various disaccharides.
Bioscience Biotechnology and Biochemistry 04/2009; 73(3):671-6. · 1.28 Impact Factor
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ABSTRACT: A beta-D-glycosidase (G I) from the China white jade snail showed non-Michaelis-Menten mode in catalyzing the reaction using pNPGlu and pNPFuc as the substrate and monitoring the released pNP. We determined quantitatively both the transglycosidic and hydrolytic products of pNPGlu and pNPFuc solvolysis for the detailed kinetic analysis on G I-catalyzed hydrolysis and transglycosylation reaction. The inhibition kinetic studies using deoxynojirimycin (DNJ) and butanol as inhibitors were preceded. DNJ only inhibited competitively the hydrolysis of cellobiose and pNPGlu while "activated" the transglycosylation of pNPGlu and pNPFuc. This was evident from the increased V(max)tr value with no change of the apparent K(m)tr. In contrast, butanol exhibited a competitive inhibition to the transglycosylation reaction and non-competitive inhibition to the hydrolysis. The results indicated that the non-Michaelis-Menten kinetic behavior was caused by the co-occurrence of substrate transglycosylation reaction. This study provided a simple method to increase the transglycosylation yield by using DNJ to inhibit hydrolysis.
Journal of Biotechnology 01/2009; 139(3):229-35. · 3.05 Impact Factor
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ABSTRACT: A rapid and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method was developed for the qualitative and quantitative determination of UGT2B7 activity using 3'-azido-3'-deoxythymidine (AZT) as probe substrate in human liver microsomes (HLMs). The method was validated for the determination of AZT glucuronidation (AZTG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C18 column (50 mm x 2.1mm i.d., 1.7 microm), with phase of acetonitrile-water (ratio 6:94). Selective ion reaction (SIR) monitor was specific for AZT, AZTG and I.S. The method was linear over the concentration range 0.5-500 microM for AZTG in spiked HLMs. Good precision and accuracy were obtained for concentrations over the standard curve range. AZTG was stable at 4 degrees C for at least 72 h in spiked liver microsomes samples. The method was successfully used to determine the kinetics of UGT activities toward AZT in HLMs. In addition, the method could determine the effects of fluconazole, a known UGT2B7 selective inhibitor, on AZTG in HLMs. Therefore, this method is suitable for in vitro studies using AZTG formation as an index reaction for UGT2B7 activity.
Journal of Chromatography B 08/2008; 870(1):84-90. · 2.89 Impact Factor
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ABSTRACT: Species delimitation in Taxus has been controversial and it is very difficult to distinguish yew materials by their morphological characters. In this paper, a valid HPLC fingerprinting method coupled with multivariate analysis was used to define a framework for Taxus species identification and classification. Fingerprint-based similarity was employed for a chemotaxonomic study by hierarchical clustering analysis (HCA) and principal component analysis (PCA). Based on the PCA loadings, twelve chemical constituents were selected as chemotaxonomic markers which can be used to establish a more practical classification. Finally, eight studied species could be divided into six well-supported groups and most samples can be assigned to the correct species. Additionally, twelve markers were tentatively identified by LC/MS.
Planta Medica 07/2008; 74(7):773-9. · 2.15 Impact Factor
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ABSTRACT: A beta-D-xylosidase from Leifsonia shinshuensis DICP 16 was purified to apparent homogeneity using a combination of ammonium sulfate precipitation, DE 52 anion-exchange, Q-Sepharose Fast Flow anion-exchange, Toyopearl Butyl 650C hydrophobic-interaction and Sephacryl S-300 HR gel-permeation chromatography. The purified xylosidase consisted of two same subunits and had the relative molecular weight of 180 kD as determined by SDS-PAGE and gel-permeation chromatography. The maximal beta-D-xylosidase activity occurred at 55 degrees C and pH 7.0. It was stable at 45 degrees C and retained its original activity for 60 min. The stability declined rapidly when the temperature rose above 55 degrees C. The xylosidase was stable in the pH range from 6.0 to 11.0 for 20 h. At pH 7.0 and 45 degrees C the Km for p-nitrophenyl-beta-D-xylopyranoside (pNPX) was 1.04 mmol/L and the Vmx was 0.095 mmol nitrophenol/min/mg xylosidase. The enzyme was inhibited strongly by Fe2+ and Cu2+. It exhibited low levels of activity against other artificial substrates, compared to its activity against pNPX. When different natural xylosides were used as the substrates, the xylosidase showed distinct hydrolysis ability. It could hydrolyze 20-C, beta-(1-->6)-xyloside of ginsenoside Rb3 (G-Rb3) into ginsenoside Rd, but did not hydrolyze the other beta-D-glucosidic bonds of G-Rb3. Additionally, the xylosidase could not hydrolyze C-7 xylosyl-bearing taxanes.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 06/2008; 24(5):867-73.
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ABSTRACT: A highly stable β-glucosidase from the viscera of China white jade snail was purified to apparent homogeneity. This purified glucosidase consisted of two identical subunits with a native molecular mass of approximately 230 kDa. The maximal activity to p-nitrophenyl-β-d-glucopyranoside (pNPG) occurred at 70 °C and pH 5.6. Its most notable characteristic was stable over a wide pH range (3–11 at 30 °C for 24 h) and a relatively high temperature (60 °C for 24 h). The Km for pNPG was 0.338 mM and the Vmax was 0.25 mmol nitrophenol/min/mg glucosidase at pH 5.6 and 50 °C. Compared to its activity against pNPG (100%), the β-glucosidase exhibited low levels of activity against other aryl-glycosides (less than 1%). Additionally, the enzyme hydrolyzed the 20-C, β-(1 → 6)-glucoside of ginsenoside Rb1 to produce ginsenoside Rd, but did not hydrolyze the other β-d-glucosidic bonds of Rb1. The properties of the enzyme could make it become a useful tool in biotransformation of glucosides.
Process Biochemistry.
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ABSTRACT: A broad specificity β-d-glycosidase, designated G I, was purified to homogeneity from the viscera of the China white jade snail (Achatina fulica). The enzyme was a monomeric protein with molecular weight of 115 kDa. G I has broad glycone specificity towards p-nitrophenyl derivatives of β-d-glucose, β-d-fucose, β-d-galactose, α-d-glucose and some disaccharides. The optimum pH and temperature are 5.5 and 55 °C, respectively. It was stable over a wide pH range (5–10 at 30 °C for 24 h) and against a relatively high temperature (50 °C for 4 h). Moreover, it was also stable and active in the presence of various alcohols. With pNPGlu, pNPFuc and cellobiose as donor, G I showed high transglycosylation activity. Six transglycosylation products were isolated from the reaction mixture containing 20% alcohol as glycoside acceptor using a preparative thin layer chromatography (preparative TLC) and identified as alkyl-glucosides and alkyl-fucosides by mass spectrometry (MS) analysis. Combining the high alcohol tolerance, moderate temperature and pH stability and alkyl glycosides production efficiency through transglycosylation, G I can be considered to be a promising candidate for the production of various alkyl glycosides.
Enzyme and Microbial Technology.
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ABSTRACT: A β-d-glucosidase, with broad regiospecific activity and designated as G II, was purified to homogeneity from the acetone power of viscera of the China white jade snail (Achatina fulica). G II consisted of two identical subunits (110 kDa) with a native molecular mass of 220 kDa. G II was stable over a wide pH range (4–10 at 30 °C for 24 h) and at a relatively high temperature (50 °C for 8 h). Moreover, it can cleave both β-(1 → 2)-glucosidic linkage at 3-C and β-(1 → 6)-glucosidic linkage at 20-C of ginsenosides and can hydrolyze ginsenosides Rb1, Rb2, Rb3 and Rc into their active metabolites, compound K, compound Y, Mx, and Mc, respectively. The optimal activity against p-nitrophenyl-β-d-glucopyranoside (pNPG) was at 50 °C and pH 5.0. Under the same condition, the Km for pNPG was 0.224 mM with a Vmax of 0.203 mmol nitrophenol min−1 mg−1. Of the substrates tested, G II specifically hydrolyzed the β-d-glucosides involving aryl-β-glucosides, alkyl-β-glucosides, and β-linked disaccharides (i.e. sophorose, gentiobiose, and cellobiose). These findings make the novel enzyme potentially useful in biotransformation processes as well as in the modification of multiple ginsenosides.
Enzyme and Microbial Technology.
