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In this study, we studied the biochemical characterization of flavone synthase I from Daucus carota (DcFNS I) and applied it with flavonoid 6-hydroxylase from Scutellaria baicalensis (SbCYP) to convert flavanones to flavones. The recombinant DcFNS I was expressed in the form of the glutathione-S-transferase fusion protein. Rather than taxifolin, naringenin, pinocembrin, and eriodictyol were accepted as substrates. The optimal temperature and pH for reaction in vitro were 35 °C and 7.5, respectively, and 2-oxoglutarate was essential in the assay system. Co

Pseudoionone is a valuable intermediate for the flavor industry. However, few attempts have been made to synthesize pseudoionone using biotechnology. In this work, an Escherichia coli strain harboring both the isopentenol utilization pathway (IUP) and mevalonate (MVA) pathway was engineered for the production of the pseudoionone precursor lycopene, which increased the titer of lycopene to approximately 20-fold the yield obtained by the original MEP pathway. Subsequently, the crucial limiting step of carotenoid cleavage dioxygenases (CCDs) was evaluated and optimized. The most effective upstream module for pseudoionone synthesis in E. coli was determined to be MnCCD1 from Morus notabilis without a GST-tag and under the control of the tac promoter. Finally, the highest pseudoionone production achieved 20.61 mg/L under the optimum fermentation conditions in a shake flask. This study provided an efficient approach for pseudoionone production and advanced our understanding of the characteristics of the CCD family for the biosynthesis of aroma compounds.

Synthesis of β-ionone in microbial cell factories is limited by the efficiency of carotenoid cleavage dioxygenases (CCDs). To obtain genes responsible for specific cleavage of carotenoids generating β-ionone, a novel carotenoid cleavage dioxygenase 1 from Morus notabilis was cloned and overexpressed in Escherichia coli . The MnCCD1 protein was able to cleave a variety of carotenoids at the positions 9, 10 (9', 10') to produce β-ionone, 3-hydroxy-4-oxo-β-ionone, 3-hydroxy-β-ionone, and 3-hydroxy-α-ionone in vitro . MnCCD1 could also cleave lycopene and β-carotene at the 9, 10 (9', 10') bind bond to produce pseudoionone and β-ionone respectively in E.coli accumulating carotenoids. The enzyme activity of MnCCD1 was reached 2.98 U/mL at optimized conditions (temperature 28 ℃, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of MnCCD1 revealed the optimal activities were at pH 8.4 and 35 ℃. The addition of 10% ethanol could increase enzyme activity at above 15%. However, an obvious decline was observed on enzyme activity as the concentration of Fe 2+ increased (0-1 mM). The V max for β-apo-8'-carotenal was 72.5 U/mg, while the K m was 0.83 mM. The results provide a foundation for developing the application of carotenoid cleavage dioxygenases as biocatalysis and synthetic biology platforms to produce volatile aroma components from carotenoids.

A sensitive, accurate, simple, and rapid analytical UHPLC-MS/MS method was developed for identification and quantification of koumine, gelsemine, and gelsenicine in human hair. Approximately 10 mg of hair was extracted with methanol by cryogenic grinding. The limits of detection (LODs) ranged from 1 to 5 pg/mg, and the limits of quantitation (LOQs) ranged from 2 to 10 pg/mg. The method was linear over a concentration range from the LOQs to 1000 pg/mg, and the linear correlation (R

A sensitive, accurate, simple, and rapid analytical LC–MS/MS method was developed for the identification and quantification of koumine, gelsemine, and gelsenicine in human hair. Approximately 10 mg of hair was extracted with methanol by cryogenic grinding. The limits of detection (LODs) ranged from 1 to 5 pg/mg, and the limits of quantitation (LOQs) ranged from 2 to 10 pg/mg. The method was linear over a concentration range from the LOQs to 10000 pg/mg, with the linear correlation (R2) of the calibration curves for all analytes above 0.998. The bias varied from -3.6 to 8.8%, while the intra- and inter-day precision Relative Standard Deviation (RSD) values ranged from 1.8–10.8% and 1.9–9.6%, respectively. Recoveries were within the range of 77.8% to 83.3%, and matrix effects were in the range of 74.3% to 87.4%. The present method was suitable for quantitative determination of koumine, gelsemine, and gelsenicine in human hair from one Gelsemium elegans poisoning case. The highest concentrations of koumine, gelsemine, and gelsenicine in the hair segment corresponding to the period of ingestion were 29.4, 17.6, and 4.7 pg/mg, respectively. To our knowledge, this study is the first to describe hair analysis in a Gelsemium poisoning case and to provide quantitative toxicological findings.

This paper describes a method for the simultaneous quantitation of risperidone and its major metabolite, 9-hydroxyrisperidone, in beagle dog plasma by field-amplified sample injection in capillary zone electrophoresis. The separation was carried out at 25°C in a 48 cm × 75 μm fused-silica capillary with an applied voltage of 20 kV using 60 mM NaH2 PO4 buffer (pH 3.6). The detection wavelength was 280 nm. Clean-up and preconcentration of plasma samples were conducted by 96-well formatted liquid-liquid extraction. In this study, this stacking technique provided a sensitivity enhancement of approximately 158-188 folds compared with the same sample without stacking. The method was suitably validated with respect to stability, specificity, linearity, lower limit of quantitation, accuracy, precision, and extraction recovery. Calibration curves exhibited good linearity (r2 > 0.995) over a wide concentration range of 2.5-200 ng/ml for both risperidone and 9-hydroxyrisperidone. The intra- and inter-day precisions at the three quality control levels were less than 11.40%. The intra- and inter-day accuracies ranged from 87.90% to 107.17% for risperidone and from 88.43% to 105.92% for 9-hydroxyrisperidone. All validation data were within the required limits. In conclusion, the method developed was successfully applied to pharmacokinetic studies of risperidone and 9-hydroxyrisperidone in beagle dogs. This article is protected by copyright. All rights reserved.

A novel and potent anti-tuberculosis drug combination pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO), and clofazimine (CFZ) that rapidly kills Mycobacterium tuberculosis (Mtb) in the lungs has been identified using the artificial-intelligence-enabled parabolic response surface approach. A universal and highly sensitive two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method for the simultaneous determination of PZA, EMB, PTO, and CFZ in various biological samples in different states (liquid samples: plasma, bile, and urine; solid samples: tissue and feces) using simple pretreatment was established and validated. For the first dimension of this column-switching arrangement, the automated purification and enrichment of the drugs were achieved on a Polar-RP column. The subsequent analytical separation was performed on an Agilent Zorbax SB-Aq column, and the total loop time was 7.5 min. The positive-ionization mode with multiple reaction monitoring was used for detection. The sensitivity was good with no carry-over detected, and the lower limit of quantification ranged from 100 to 500 pg/mL. This quantification method was fully validated and proved to be robust in accordance with US Food and Drug Administration guidelines. High recoveries (85.3-111.4%) and accuracies (92.1-109.3%), together with high precision values (0.5-13.8%), were verified in all matrices. All standard curves showed favorable linearities with r2 > 0.995. This validated method was applied to study plasma pharmacokinetics, tissue distribution, and excretion in Sprague-Dawley rats after oral administration of the drug combination.