Search

2-ketoglutarate (2-KG)/Fe 2+ -dependent dioxygenases can catalyze the highly specific regio- and stereoselective functionalization of C(sp 3 )-H bond of complex compounds under mild reaction conditions. Hyoscyamine 6β-hydroxylase (H6H), a member of these dioxygenases, catalyzes two consecutive oxidation reactions in the synthesis of scopolamine. The first reaction is the hydroxylation of hyoscyamine to 6β-hydroxyhyoscyamine and the second is epoxidation of 6β-hydroxyhyoscyamine. This paper introduces the catalytic mechanism, substrate scope, and application of H6H and evaluates the possibility of this enzyme as a biocatalyst for the functionalization of C(sp 3 )-H bond in complex compounds with different structural characteristics via hydroxylation or epoxidation, providing a theoretical basis for modification and application of this enzyme.

Copyright of Bulletin of the Chinese Ceramic Society is the property of Bulletin of the Chinese Ceramic Society Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of China Pulp & Paper is the property of China Pulp & Paper Magazines Publisher and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Preparation process and property of hydroxylation modified cotton fiber base absorbent were discussed. Cotton Fibers were used as the base material. Epoxidation and dialdehyde oxidation were performed for the cotton fibers with epoxy chloropropane and sodium periodate. Then, epoxy chloropropane was used as cross-linking agent to graft β-cyclodextrin and aldolization was adopted to graft hydroxy end hyperbranched polymer. Through the adsorption experiment on heavy metal ion, the optimized preparation process was obtained as 40 °C for oxidizing temperature, 0.6% for sodium periodate mass fraction and 3.5 h for dialdehyde oxidation. When 0.4 mol/L HCl was used as desorption reagent and 5% NaOH mass fraction was used as regenerant, the desorption and regeneration efficiency were better. It is considered that the absorption capability of the modified cotton fiber base absorbent on heavy metal ion is higher than cotton fiber. Through desorption of HCl and regeneration of NaOH regenerant, the absorption effect is still quite high. [ABSTRACT FROM AUTHOR]

Copyright of Modern Food Science & Technology is the property of Editorial Office of Modern Food Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

17α hydroxylase is a key enzyme for the conversion of progesterone to prepare various progestational drug intermediates. To improve the specific hydroxylation capability of this enzyme in steroid biocatalysis, the CYP260A1 derived from cellulose-mucilaginous bacteria Sorangium cellulosum Soce56 and the Fpr and bovine adrenal-derived Adx 4-108 derived from Escherichia coli str. K-12 were used to construct a new electron transfer system for the conversion of progesterone. Selective mutation of CYP260A1 resulted in a mutant S276I with significantly enhanced 17α hydroxylase activity, and the yield of 17α-OH progesterone reached 58% after optimization of the catalytic system in vitro . In addition, the effect of phosphorylation of the ferredoxin Adx 4-108 on 17α hydroxyl activity was evaluated using a targeted mutation technique, and the results showed that the mutation Adx 4-108 T69E transferred electrons to S276I more efficiently, which further enhanced the catalytic specificity in the C17 position of progesterone, and the yield of 17α-OH progesterone was eventually increased to 74%. This study provides a new option for the production of 17α-OH progesterone by specific transformation of bacterial-derived 17α hydroxylase, and lays a theoretical foundation for the industrial production of progesterone analogs using biotransformation method.

Recombinant collagen, as an alternative to natural collagen, has the potential to be widely used in biomaterials, biomedicine, etc. Diverse recombinant collagens and their variants can be industrially produced in a variety of expression systems, which lays a foundation for exploring and expanding the clinical application of recombinant collagens. We reviewed different expression systems for recombinant collagens, such as prokaryotic expression systems, yeast expression systems, as well as plant, insect, mammal, and human cell expression systems, and introduced the advantages, potential applications, and limitations of recombinant collagen. In particularly, we focused on the current progress in the recombinant collagen production, including recombinant expression system construction and hydroxylation strategies of recombinant collagen, and summarized the current biomedical applications of recombinant collagen.

Nano-LC MS/MS was used to analyze trypsin digested deer-hide gelatin(DHG) samples, hydroxylation and O-glycosylation on lysine sites of DHG were comprehensive identified by using PEAKS Studio software. The sites, sorts and amounts of hydroxylation and O-glycosylation on Type Ⅰ collagen α1 chain(COL1 A1) and α2 chain(COL1 A2) of DHG were revealed. As a result, 5 284 peptides were identified from DHG samples, which were mainly from COL1 A1 and COL1 A2. Among these peptides, there were 449 peptides with hydroxylysine, 442 with galactosyl-hydroxylysine, 449 with glucosyl-galactosyl-hydroxylysine. The major modified sites of hydroxylation and O-glycosylation in DHG were shown as follow: α1-9 N and α2-5 N in N-telopeptides, α1-87, α1-174, α1-930, α2-87, α2-174, α2-933 in triple helix domain, and α1-16 C in C-telopeptides. These hydroxylation and O-glycosylation were correlated with the formation and stability of collagen molecules and collagen fibrils. It is feasible for the collagens and peptides dissolving from deer skin collagen fibrils under high temperature and pressure decocting, high temperature and pressure also might destroy inter-molecular covalent cross-linking and help those glycol-peptides formations. The present study provided ideas and strategies for the in-depth investigation on DHG chemical constituents, and showed good theoretical significance and application value.

Hydroxy amino acids, constituents of chiral pharmaceutical intermediates or precursors, have a variety of unique functions in the research fields of biotechnology and molecular biology, i.e. antifungal, antibacterial, antiviral and anticancer properties. Biosynthesis of hydroxy amino acids is preferred because of its high specificity and selectivity. The hydroxylation of hydrophobic amino acids is catalyzed by hydroxylase, which belongs to the mononuclear non-heme Fe(Ⅱ)/α-ketoglutarate-dependent dioxygenases (Fe/αKGDs). Fe/αKGDs utilize an (Fe(Ⅳ)=O) intermediate to activate diverse oxidative transformations with key biological roles in the process of catalytic reaction. Here, we review the physiological properties and synthesis of hydroxy amino acids, especially for the 4-HIL and hydroxyproline. The catalytic mechanism of Fe/αKGDs is elucidated, and the applications of hydroxy amino acids in industrial engineering are also discussed.

Perillyl alcohol, [4-isopropylene-1-cyclohexene] methanol, is a monocyclic monoterpene alcohol with special odorous similar to that of linalool and terpineol. It has application potential in pharmaceutical, daily chemical and food industries. In this study, one method for the synthesis of perillyl alcohol through the MVA pathway was studied. First, the MVA metabolic pathway originated from Enterococcus faecalis was constructed in Escherichia coli to synthesize limonene. Limonene was further transformed to perillyl alcohol by the hydroxylation of cytochrome P450 alkane hydroxylase. Furthermore, the shake flask fermentation condition of the engineered E. coli strain was optimized. The results showed that the engineered E. coli could produce about 50.12 mg/L perillyl alcohol through MVA pathway using glucose as raw material. In this study, the method of the MVA pathway for perillyl alcohol synthesis was constructed successfully in engineered E. coli, which provides both theoretical and technical support for terpenoids biosynthesis.

Erythromycin A is a clinically important macrolide antibiotic with broad-spectrum activity. Its biosynthesis involves the formation of the 14-membered skeleton catalyzed by polyketide synthases, and the modification steps such as hydroxylation, glycosylation and methylation. Based on the understanding of the biosynthetic mechanism, it is reliable to genetically manipulate the erythromycin A-producing strain for production improvement and structure modification. In this paper, we reviewed the progress regarding erythromycin A in high-producing strain construction and chemical structure derivation, to provide insights for further development.

In order to discover the steroid biotransformation ability of filamentous fungus Aspergillus niger TCCC41650, we studied the fermentation of 4-androstene-3,17-dione with A. niger TCCC41650. The transformation product was purified, crystallized and determined as 16β-hydroxy-androst-4-ene-3,17-dione by X-ray single crystal diffraction method. The best fermentation condition was found to be pH 6.0, ethanol amount 2% with a substrate concentration of 1 per thousand, the transformation rate is 85.81% after 72 h. Based on the best of our knowledge, 16β-hydroxylation rarely occurs in microbial transformations of steroid. This study laid the foundation for the research of 16β-hydroxylation steroids

Prolyl-4-hydroxylase domain (PHDs) family is one of the most important regulatory factors in hypoxic stress. PHD2 plays a critical role in cells and tissues adaptation to the low oxygen environment. Its hydroxylation activity regulates the stability and transcriptional activity of the hypoxia-inducible factor 1 (HIF-1), which is the key factor in response to hypoxic stress. Subsequently, PHD2 acts as an important factor in oxygen homeostasis. Studies have shown that PHD2, through its regulation on HIF-1, plays an important role in the post-ischemic neovascularization. Furthermore, under hypoxic condition, PHD2 also regulates other pathways that positively regulate angiogenesis factors HIF-1 independently. Moreover, recently, several evidences have also shown that PHD2 also affects tumor growth and metastasis in a tumor microenvironment. Based on these facts, PHD2 have been considered as a potential therapeutic target both in treating ischemic diseases and tumors. Here, we review the molecular regulation mechanism of PHD2 and its physiological and pathological functions. We focus on the role of PHD2 in both therapeutic angiogenesis for ischemic disease and tumor angiogenesis, and the current progress in utilizing PHD2 as a therapeutic target.

Combined with method of ketoconazole resistance screening, a 7alpha,15alpha-diOH-DHEA high-producing mutant Colletotrichum lini ST-1 was obtained by compound mutation of NTG and low energy N+ ion beam implantation. With the substrate concentration of 10 g/L DHEA, the molar yield of 7alpha,15alpha-diOH-DHEA reached 34.2%, increased by 46.2% than that of the original strain. Then we optimized the medium. First, Plackett-Burman design was used to evaluate the effects of medium components on molar yield of the product. Results show that glucose, yeast extract and MgSO4 x 7H2O were the important parameters for the biotransformation process. Subsequently, the path of steepest ascent was used to approach the optimal levels. To obtain the optimal levels, central composite design and response surface analysis were carried out. The optimal medium was as follows (g/L): glucose 26.34, yeast extract 12.15, corn flour 3.00, FeSO4 x 7H2O 0.015, MgSO4 x 7H2O 0.14, KH2PO4 0.90. Under the optimal conditions, the molar yield of 7alpha,15alpha-diOH-DHEA reached 49.3%, which was 44.2% higher than that of using the medium before optimization.

Wuxistatin, a novel and potent statin, is converted from lovastatin by Amycolatopsis sp. CGMCC1149. In the bioconversion, lovastatin is firstly hydroxylated by a hydroxylase. To obtain the critical hydroxylase, a novel hydroxylase gene was isolated from Amycolatopsis sp. CGMCC1149 by Degenerate PCR and Self-Formed Adaptor PCR and expressed in Escherichia coli. BLAST sequence analysis revealed that the gene belonged to cytochrome P450 gene superfamily and could encode a 403-amino-acid protein with a molecular weight of 44.8 kDa. The secondary structure prediction result showed that this protein contained many typical functional regions of P450, such as oxygen binding site, ion-pair region and heme binding region. Meanwhile, a catalytic function verification system was constructed by NADH, ferredoxin and ferredoxin reductase which could catalyze lovastatin hydroxylation into the target product. These would be helpful for further studies in large-scale production of wuxistatin.

Raman spectroscopy was used to study the influence of ultraviolet-A(UV-A) radiation on collagen I. The Raman spectra of collagen I and that after 90 min UV-A radiation were reported. The results proved that irradiation with 90 min UV-A caused the change in the structures of collagen I. Intramolecular hydrogen bonds were broken, and the hydrogen bonding system was changed. The intensity of helix was decreased, while the intensity of the disordered conformation in proteins such as random coil was increased. Otherwise, the UV-A radiation influenced the hydroxylation of proline and the content of hydroxyproline was reduced. The changes caused by UV-A radiation could damage the triple helical structure of collagen I. It would lead to a series of changes, such as the destruction of collagen fibers during the photoaging of skin.

Sinisan is a widely used traditional Chinese medicine (TCM) in treating various diseases; however, the in vivo metabolic profile of its multiple components remains unknown. In this paper, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was applied to identify the metabolites of Sinisan extract in rat plasma, urine, feces and bile after intragastric administration. Using MS(E) and mass defect filter techniques, 41 metabolites of 10 parent compounds (naringin, naringenin, hesperidin, neohesperidin, liquiritin, liquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, saikosaponin a and saikosaponin d) were detected and tentatively identified. It was shown by our results that these compounds was metabolized to the forms of hydroxylation, glucuronidation, sulfation, glucuronidation with sulfation and glucuronidation with hydroxylation in vivo.

As a novel class of organic pollutants, OH-PBDEs are becoming a new research topic. Recently, OH-PBDEs correlated with their bioaccumulation and toxic effects have been found in various environmental matrices. This paper reviews recent researches about the source, determination, exposure level, toxicological effects and transformation pathways of OH-PBDEs. Furthermore, the recommendations for further research about OH-PBDEs are also proposed in this paper.

Objective: Puerarin is a Chinese herbal medicine. We isolated a fungus from soil which could convert puerarin to 3'-hydroxypuerarin., Methods: Strains were cultivated on potato dextrose agar (PDA). The biotransformation product was extracted with an organic solvent and detected on high performance liquid chromatography(HPLC)., Results: A total of 186 fungal strains were isolated from soil, and strain NT-01 was determined to transform puerarin into 3'-hydroxypuerarin. The product was identified as 3'-hydroxypuerarin by MS and 1H-NMR and 13C-NMR. The morphology characteristic of strain NT-01 was consistent with Gliocladium deliguescens. Phylogenic analysis on sequences showed that strain NT-01 was clustered together with G. deliguescens and G. viride with high boostrap supporting, so that strain NT-01 was identified as Gliocladium sp., Conclusion: Gliocladium strain NT-01 can convert puerarin to 3'-hydroxypuerarin.

Flow-injection chemiluminescence was applied to determine the trace polyhydroxy phenol in environment (soil and water). Based on the comparison of several different chemiluminescence systems and optimization, an alkaline luminol-NaIO4 system for determinating polyhydroxy phenol was chosen. During the process of polyhydroxy phenol determination, the effect of dissolved humic acid (HA) on the determination of phenol by this chemiluminescence system was also considered, which generally coexists in soil and water widely. Thus the present work provided the reference for the determination of the real samples coming from soil or water. An obvious restraining effect of humic acid on the chemiluminescence signal obtained by the luminol-NaIO4-polyhydroxy phenol system was found, which shows a linear relationship with the concentration of HA in a certain range, the linear equation is y = 70.36x + 540.1, the correlation coefficient is 0. 9954, the linear range is between 3-15 mg x L(-1), the limitation of detection is 0.749 mg x L(-1), and the relative standard deviation (RSD) is 1.08% when the concentration of HA is 6 mg x L(-1). The possible working mechanism of humic acid here is discussed and deduced.

Objective: To investigate the interaction between hypoxia-inducible factors-1alpha subunit (HIF-1alpha) and its three prolyl hydroxylases (PHD1, PHD2 and PHD3) during the development of rat hypoxic pulmonary hypertension., Methods: Forty male SD rats were randomly divided into 5 groups and exposed to normoxia (C group) or exposed to hypoxia for 3, 7, 14 or 21 d (H(3), H(7), H(14), H(21) group), respectively. Mean pulmonary arterial pressure (mPAP), vessel morphometry and right ventricle hypertrophy index (RVHI) were measured. Reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization were used to determine the expression of mRNA. Immunohistochemistry and Western blot were used to determine the expression of mRNA., Results: The level of mPAP [(21.7 +/- 2.4) mm Hg, 1 mm Hg = 0.133 kPa], the ratio of vascular wall thickness to external diameter [WA%, (43.9 +/- 5.3)%] and pulmonary artery media thickness [PAMT, (10.0 +/- 0.7) microm] were significantly higher in H(7) group than those in C group [(16.6 +/- 1.6) mm Hg, (36.3 +/- 4.8)% and (8.5 +/- 1.3) microm respectively, q value were 5.591, 4.082, 2.929, respectively, all P < 0.05]. These parameters reached a high level and remained stable on H(14) group, and RVHI was significantly higher in H(14) group [(27.6 +/- 1.4)%] than in C group [(23.6 +/- 2.9)%, q = 5.817, P < 0.05]. HIF-1alpha protein was barely positive in C group (0.080 +/- 0.009), but markedly up-regulated in H(3) group (0.196 +/- 0.018, compared with C group q = 18.864, P < 0.05), reaching its peak in H(7) group (0.203 +/- 0.022), and then declined slightly in H(14) and H(21) group. HIF-1alpha mRNA increased marginally in H(14) group (0.176 +/- 0.019, compared with C group q = 5.401, P < 0.05, 0.139 +/- 0.017). PHD1 and PHD2 mRNA (0.260 +/- 0.031, 0.196 +/- 0.023) and protein (0.244 +/- 0.030, 0.205 +/- 0.025) were positive in C group. PHD2 mRNA and protein were up-regulated in H(3) group (0.246 +/- 0.023, 0.235 +/- 0.025, compared with C group q value was 5.268, 3.046, respectively, all P < 0.05), reaching its peak in H(14) group whereas PHD1 protein declined in H(14) group (0.210 +/- 0.023, compared with C group q = 3.885, P < 0.05) without significant mRNA change. PHD3 mRNA and protein were detected at low level in C group (0.110 +/- 0.013, 0.153 +/- 0.019), but markedly up-regulated in H(3) group (0.259 +/- 0.024, compared with C group q = 15.831, P < 0.05), and then PHD3 mRNA remained at high level while PHD3 protein declined in H(14) and H(21) group (0.206 +/- 0.025, 0.189 +/- 0.019, compared with H(7) group q value was 6.441, 8.526, respectively, all P < 0.05). Linear correlation analysis showed that HIF-1alpha mRNA and protein were positively correlated with mPAP. There was a positive correlation between HIF-1alpha protein and PHD2, PHD3 mRNA (r value was 0.580, 0.690, respectively, all P value was 0.000) but a negative correlation between HIF-1alpha protein and PHD2 protein (r = -0.704, P < 0.05)., Conclusions: HIF-1alpha was regulated mainly at the protein level during the development of hypoxic pulmonary hypertension. PHD2 and PHD3 are inducible by hypoxia, possibly via elevated HIF-1alpha, suggesting that a hypoxic up-regulation of PHD acts via feedback mechanism to attenuate hypoxia induced responses. PHD may also be regulated by posttranscriptional mechanisms.

Aim: To identify the hydroxylate metabolites and its sulfate conjugates of daidzein in rat urine., Methods: Urine samples from 0 - 24 h were collected after single ig dose of 500 mg x kg(-1) daidzein to each of six rats. The urine samples were purified by SPE column (SPE C18) and analyzed with liquid chromatographic-tandem electrospray ionization ion trap mass spectrometry (LC-ESI/MS(n)) for potential metabolites., Results: Several new hydroxylate metabolites and its sulfate conjugates were found and identified in rat urine., Conclusion: LC-ESI/MS(n) is proved to be a simple, rapid, sensitive and specific technique for identification of the hydroxylate metabolites and its sulfate conjugates of daidzein in rat urine.

A strain named NJ2 was isolated from the soils in Nanjing area, and its resting cells transformed IMI into a polar product. The strain was identified as Stenotrophomonas maltophilia based on BioMerieux Vitek system and the analysis of 16S rDNA sequence. Mass spectrometry of the product extracted with an organic solvent and re-crystallization gave m/z value of 272 for the molecular ion while 256 for substrate IMI. It demonstrated that the transformed product was the hydroxylated derivate of IMI or hydroxy IMI. NMR analysis further indicated that the hydroxyl moiety located at the ring of imidazolidine and the transformed product was 5-hydroxy IMI. Kinetics of transformation IMI showed that the content of IMI decreased 1.15 mmol/L and the content of transformed product 5-hydroxy IMI increased 1.10 mmol/L after transformation for 10 days. The molar conversion yield was 95.9%. The S. maltophilia NJ2 strain, with the characterization of strong transformation ability and high molar conversion yield, can be used in industrial production of 5-hydroxy IMI for synthesis of highly insecticidal olefin IMI.

The 11-hydroxylation of 16alpha,17alpha-epoxy-4-pregenene-3,20-dione as a useful intermediate for the preparation of hormones can be achieved by the mycelium of Absidia coerulea at higher conversion rate than using other strains. In this paper 16alpha,17alpha-epoxy-4-pregenene-3,20-dione mixed with a little water, beta-cyclodextrin, Tween-80 was introduced into the fermentation broth after ultrasonication to increase pseudo-water-solubility of the hydrophobic substrate. This pseudo-crystallo feed could avoid the toxicity of organic solvents and was more available for the microbial transformation. The multi layer feed-forward neural network was used to setup a model which indicated the relationship between medium and feed components and the conversion rate. Particle swarm optimization (PSO), which was a stochastic global optimization algorithm and of which the convergence speed was high, was applied to obtain the optimal concentration of the medium and feed components. At optimum conditions with the pseudo-crystallo feed, the conversion rate of 16alpha,17alpha-epoxy-4-pregenene-3,20-dione at an initial concentration of 10 g/L was 87.5% in shaking flasks. The conversion rate of the substrate was up to 86.6% at higher concentration of 20 g/L feed in a 3.7 L fermentor.

Aim: To identify the drug-metabolizing enzymes involved in the hydroxylation of the new anti-inflammatory and anodyne imrecoxib., Methods: Imrecoxib was incubated with heterologous expression human cytochrome P450 (rCYPs) in vitro, and metabolites and remained parent drug were detected with liquid chromatography-multistage mass spectrometry. The contribution of 4 CYPs in the hydroxylation metabolism of imrecoxib was evaluated by total normalized rate (TNR) method., Results: Imrecoxib is metabolized by CYP2C9, CYP2D6 and CYP3A4, with the rate of 62.5%, 21.1% and 16.4%, respectively., Conclusion: CYP2C9 is the major enzyme involved in imrecoxib hydroxylation metabolism.

Some features of Pseudomonas putida NA-1 strain in cultivation and enzyme production were different from S. marcescens IFO 12648 and P. fluorescens TN5 stains which could transform nicotinic acid to 6-hydroxynicotinic acid reported by other scientists, such as optimal carbon and its optimal concentration, optimal inducer concentration, and optimal cultivation temperature. The ideal transformation condition was nicotinic acid 3%, temperature 35 degrees C and pH 7.0. Under an appropriate condition, in a 4 liter fermenter, production yield of 6-hydroxynicotinic acid by Pseudomonas putida NA-1 was 108.39 g/L.

Strain screening was conducted on a large scale in order to develop a biotransforming process for 6-hydroxynicotinic acid production. A strain named as NA-1 was isolated from soil which could transform nicotinic acid into a new product showed by HPLC. The new product was identified as 6-hydroxynicotinic acid by MS and 1H-NMR. The morphology, physiological and biochemical characteristics of strain NA-1 were studied and showed that characteristics of strain NA-1 were essentially consistent with Pseudomonas putida. The analysis of 16S rDNA sequence from strain NA-1 suggested that strain NA-1 was clustered together with P. putida in phylogenetic tree and the sequence identity between stain NA-1 and Pseudomonas putida was 99%. So strain NA-1 was identified to P. putida.

Aim: To identify the cytochrome P450 (CYP) isoform (s) involved in daidzein mono-hydroxylated metabolites using human liver microsomes., Methods: Kinetic analysis of the rates of formation of mono-hydroxylated metabolites of daidzein, including 7,8,4'-trihydroxyisoflavone (7,8,4'-THI), 7,3',4'-trihydroxyisoflavone (7,3',4'-THI) and 6,7,4'-trihydroxyisoflavone (6,7,4'-THI), was performed using human liver microsomes (HLM) and recombinant enzymes at substrate concentrations ranging from 0.5 to 400 micromol x L(-1). Nine selective inhibitors or substrate probes specific for different CYP isoforms were applied for screening the isoform(s) responsible for mono-hydroxylated metabolism of daidzein., Results: Michaelis-Menten kinetic parameters were best fitted to one-component enzyme kinetic model. The mean Km (micromol x L(-1) ) and V(max) (micromol x g(-1) x min(-1)) values were 27 +/- 10 and 4. 8 +/- 2.1, 54 +/- 22 and 2.3 +/- 1.0, 51 +/- 29 and 2.2 +/- 0.8, for the formation rates of 7,8,4'-THI, 7,3',4'-THI, and 6,7,4'-THI, respectively. Furafylline, the CYP1A2 specific inhibitor, estrogen, and monoclonal antibody raised against human CYP1A2 (MAB-1A2) apparently inhibited the formation of mono-hydroxylated metabolites, The IC50 of Fur for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI was 1.0, 0.9 and 0. 8 mol x L(-1), respectively. The IC50 of estrogen for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI were 51, 60 and 64 mol x L(-1) respectively. The IC50 of MAB-1A2 for the formation of the mono-hydroxylated products was 1 mol x L(-1), but neither other selective inhibitor nor substrate probes, including coumarin (CYP2D6), sulphaphenzole ( CYP2C9/10), omeprazole ( CYP2C19), quinidine (CYP2D6), diethyldithiocarbamate (CYP2E1), troleandomycin (CYP3A4) and keteconazole (CYP3A4), did so with human liver microsomes., Conclusion: The in vitro studies indicated that daidzein mono-hydroxylated products were principally metabolized by CYP1A2 in human.

Aim: To investigate the enzyme kinetics of (-)3S,4R,5R,6S-clausenamide[(-)-Clau] and (+)-3R,4S,5S,6R-clausenamide[(+)-Clau] catalyzed by rat liver microsomes and compare their stereoselective differences., Methods: An in vitro metabolic system was built by using rat liver microsomes and NADPH-generating system. Clau and its metabolites were determined simultaneously by a reversed-phase high performance liquid chromatography. The kinetic parameters, K(m), Vmax, and metabolic rate, Vmax/K(m), were calculated by Eadie-Hofstee plot., Results: In the metabolic system, (-)-Clau was found to be mainly metabolized to 7-hydroxy-, 5-hydoxy- and 4-hydroxy-Clau, and 7-hydroxylation was a preferential pathway which exhibited higher Vmax/K(m) value (0.135 microL.min-1.mg-1) than those of 5- and 4-hydroxylation (0.063 and 0.068 microL.min-1.mg-1, respectively). For (+)-Clau, it was mainly metabolized to 4-hydroxy-Clau, whereas 7-hydroxy- and 4-hydroxy-Clau were so small that they could not be detected systematically. 4-Hydroxylation of (+)-Clau showed highest Vmam/K(m) value (0.547 microL.min-1.mg-1) among all the metabolites tested, which was 8.0 times higher than that of 4-hydroxylation of its antipode., Conclusion: The data indicated that there were obvious substrate stereoselective differences in the hydroxylation metabolism of (+)- and (-)-Clau, which provided an explanation of the difference of pharmacokinetic characteristics of Clau enantiomers in rats.

Benzoic acid with weak fluorescence may react on .OH, and products with intense fluorescence are made. Extractives of Chinese traditional medicine may eliminate .OH in solution, and make amounts of the products to reduce. Then, increase level of fluorescence of products in solution will be lowered. Based on this principle, a new method is developed to determine eliminating ratio of Chinese traditional medicine for .OH. It is shown that productivity of .OH tends to saturation when H2O2 is shown more than 20 min by 280 nm UV light; .OH may react on benzoic acid completely when molar ratio of H2O2 and benzoic acid is 30:1; linear response range of products fluorescence is 2.2-80 mmol.L-1 with concentration of H2O2. IC50 of elimination .OH with magnoliae and eucommia are 1.025 and 515.3 mg.L-1 respectively. There are no remarkable difference between these results and that of spectrophotometry.

High performance liquid chromatography (HPLC) with fluorescence detector was used to determine salicylic acid (SA) and its main hydroxylated products, 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). Mobile phase was 30 mmol/L acetate(pH 4.9) and the flow rate was 1.0 mL/min. Low detection limits, 10 nmol/L for 2,3-DHBA, 0.6 nmol/L for 2,5-DHBA and 1.0 nmol/L for SA were obtained. The photolysis system of salicylic acid and H2O2 was investigated. The products in the system were measured and the yields were 45% and 51% for 2,3-DHBA and 2,5-DHBA, respectively.

Aim: To investigate the hydroxylation process of benproperine in humans., Methods: After an oral administration of 60 mg benproperine to ten healthy male volunteers, urine samples collected within 0-24 h were extracted by solid phase extraction and analyzed by liquid chromatography-ion trap mass spectrometry. A microbial transformation of benproperine combined with semi-preparative HPLC was used to get two reference substances of the hydroxylated metabolites, and their structures were then elucidated by NMR. Furthermore, the structures of conjugated metabolites were speculated based on their characteristics in MS fragmentation., Results: Five hydroxylated metabolites of benproperine and some of their conjugates with endogenous glucuronic acid or sulfuric acid were found in urine of volunteers after the dose. The structures of two metabolites were identified as 4"-hydroxybenproperine and 4"'-hydroxybenproperine by comparison of HPLC retention times and mass spectra with those of authentics obtained from the microbial transformation., Conclusion: Hydroxylation of benproperine occurs preferentially at para-position of the alkoxyl group in the aromatic ring. The hydroxylated metabolites of benproperine in human urine mainly exist as their glucuronic acid or sulfuric acid conjugates.

Two kinds of micro-organism, Arthrobacter sp. AX86(1,4-dehdrogenator) and Absidia sp. A28(11 alpha-hydroxylator) were used in this experiment. Two different fermentation techniques were performed to accomplish the multiple conversional reactions for producing 16 beta-methyl-11 alpha,17 alpha,21-trihydroxy-1,4-pregnadiene-3,20-dione(III) from 16 beta-methyl-3 beta,17 alpha,21-trihydroxy-5 alpha-pregnane-20-one-21-acetate(I): 1) To produce product(III) by means of a two-step fermentation method which were independently performed first by Arthrobacter and next by Absiaia, and 2) the product was obtained by a sequential fermentation system of aforesaid two micro-organisms in a single fermentor without isolation of the intermediates from the mixture. Our results showed that in both fermentation systems high yield of product was obtained. However, according to the technical simplicity, shorter duration of fermentation cycle and efficient yield of product, the second method is better than the first one.

Infrared spectroscopy of native and metalbinding duckweed was compared. After binding metals, the absorbance peak around 3,400 cm-1, which reflects OH group stretching vibration, had been moved about 83 cm-1, and its absorbance was decreased. The absorbance of the dominating bands near 1,650 and 1,540 cm-1, the characteristic IR absorption of protein, was decreased. Therefore, the peak absorbance at 620 cm-1 was increased significantly by exposure to high concentration of ZnSO4 solution. So we concluded that OH group play a important role in binding zinc cation and the structure of cell walls had changed due to zinc existing. Furthermore, the process of binding metals by duckweed is physical adsorption and chemical adsorption simultaneously.

An Absidia sp. 28 strain was shown to have higher activity of 11 alpha-hydroxylation using 16 beta-methyl-17 alpha,21-dihydroxy-1,4-pregnadiene-3,20-dione as a substrate. It was found that 21-acetylization of substrate could increase 11 alpha-hydroxylation rate conspicuously. The yield of the 11 alpha-hydroxylation by this strain was 73% in the conversion of 0.5% concentration of 21-acetated substrate under optimum conditions.

The possibility of involvement of cytochrome P4503A (CYP3A) in the monohydroxylation of the ring A of praziquantel (PZQ) was studied by using CYP3A specific inducer dexamethasone (DEX), specific inhibitor triacetyloeandomycin (TAO) and the activity of erythromycin (ERY) and ethylmorphine (EMP) N-demethylase which are known to be marker for CYP3A enzyme activity as probes. In the liver microsomes obtained from rats pretreated with CYP3A inducer DEX with TAO treatment the content of uncomplexed P450 was decreased, the activity of ERY and EMP N-demethylase ws reduced, and simultaneously, the rate of formation of ring A monohydroxylate of PZQ was inhibited. Ring A monohydroxylate formation was competitively inhibited by TAO and ERY. The rates of ring A monohydroxylate formation were strongly correlated with the activity of N-demethylase of ERY and EMP. These results indicate that CYP3A is involved in the monohydroxylation of the ring A of PZQ.

The metabolism of omeprazole to its two major metabolites, hydroxyomeprazole (OH-OPZ) and omeprazole sulfone (OPZ-SFN) was studied in rat liver microsomes by a reversed phase HPLC assay. The formation of metabolites of OPZ depended on incubation time, substrate concentration, microsomal protein concentration, and was found to be optimal at pH 7.4. The Vmax and Km of OPZ hydroxylation in the rat liver microsomal preparation were 2033 nmol/(min.mg protein) and 46.8 mumol.L-1 respectively. The maximum rate of formation of OPZ-SFN (Vmax) was 187.9 nmol/(min.mg protein), with a Km value of 120.7 mumol.L-1 in rat liver microsome. Moreover, the effects of 7 drugs on OPZ metabolism were tested. The results showed that mephenytoin, benzodiazepines (DZ, NDZ, TMZ, FNZ, NZ) and papaverine caused inhibition of OPZ metabolism, among them papaverine was the only fairly strong inhibitor.

The demethylation and hydroxylation of amitriptyline were calculated from the ratios between the area under concentration--time curve (AUC) of amitriptyline and its three metabolites in eight healthy Chinese volunteers after a single oral dose of 100 mg amitriptyline. Great interindividual differences in AUCs of amitriptyline and its metabolites were observed. HPLC method was used to determine the debrisoquine hydroxylation phenotype in seven out of the eight volunteers. Six subjects were found to be rapid and one slow debrisoquine hydroxylators. The ratio between debrisoquine and 4-hydroxydebrisoquine in urine correlated significantly with the rate of amitriptyline hydroxylation and the AUCs of amitriptyline and 10-hydroxyamitriptyline, but not with that of amitriptyline demethylation. There also was a weak correlation between total plasma clearance and the hydroxylation of debrisoquine. These data suggest that the hydroxylation of amitriptyline and debrisoquine may be regulated by similar enzymatic processes and the demethylation and hydroxylation processes in amitriptyline metabolism appear to undergo two separate pathways.

The metabolic ratio of debrisoquine hydroxylation (MR) determined as the ratio of debrisoquine over 4-OH-debrisoquine in 8 h urine after a single oral dose of DB (10 mg) in unrelated Chinese Zang and Wei volunteers was studied by using gas chromatography. The MR of 132 healthy Chinese Zang subjects ranged from 0.20 to 34.32, and that of 158 healthy Chinese Wei subjects ranged from 0.13 to 29.73. Two phenotypes were apparent in the frequency distribution histograms with an antimode at log MR = 1.10 (MR = 12.6). Two subjects were therefore identified as poor metabolizers (PMs) of DB in 132 Zang volunteers (the frequency of PMs was found to be 1.52%), and only one in 158 Wei volunteers (0.63%). Neither sex nor smoking habit affected the DB hydroxylation (P greater than 0.2). The recoveries of DB and 4-OH-DB in Zang volunteers were 19.83 +/- 10.99 and 11.57 +/- 7.04%, and in Wei volunteers they were 22.74 +/- 14.41 and 17.77 +/- 10.82%, respectively.

The conversion of 16 alpha-methyl-Reichstein's compound S 21-acetate (I) to 16 alpha-methyl-hydrocortisone (II) by the mycelium Curvularia lunata AS3.4381 was studied. Maximal 11 beta-hydroxylating activity of the mycelium was found during cultivation of the microorganism by 24h. Inhibition of the ethanol to the 11 beta-hydroxylating activity was obvious. The yield of 55.4% (II) (W/W) could be achieved during conversion of 0.15% substrate at 72 h.