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In this study, to construct the riboflavin-producing strain R1, five key genes, ribA, ribB, ribC, ribD, and ribE, were cloned and ligated to generate the plasmid pET-AE, which was overexpressed in Escherichia coli BL21. The R1 strain accumulated 182.65 ± 9.04 mg/l riboflavin. Subsequently, the R2 strain was constructed by the overexpression of zwf harboring the constructed plasmid pAC-Z in the R1 strain. Thus, the level of riboflavin in the R2 strain increased to 319.01 ± 20.65 mg/l (74.66% increase). To further enhance ribB transcript levels and riboflavin production, the FMN riboswitch was deleted from E. coli BL21 with CRISPR/Cas9 to generate the R3 strain. The R4 strain was constructed by cotransforming pET-AE and pAC-Z into the R3 strain. Compared to those of E. coli BL21, the ribB transcript levels of R2 and R4 improved 2.78 and 3.05-fold, respectively. The R4 strain accumulated 437.58 ± 14.36 mg/l riboflavin, increasing by 37.17% compared to the R2 strain. These results suggest that the deletion of the FMN riboswitch can improve the transcript level of ribB and facilitate riboflavin production. A riboflavin titer of 611.22 ± 11.25 mg/l was achieved under the optimal fermentation conditions. Ultimately, 1574.60 ± 109.32 mg/l riboflavin was produced through fed-batch fermentation with 40 g/l glucose. This study contributes to the industrial production of riboflavin by the recombinant E. coli BL21. [ABSTRACT FROM AUTHOR]

A rapid, simple and sensitive ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed and validated for the determination of hederasaponin B, an active triterpenoid saponin widely existed in Hedera helix L. Plasma samples were processed by protein precipitation with acetonitrile and separated on a Thermo Hypersil GOLD C18 (2.1 mm × 50 mm,1.9 μm) at flow rate of 0.3 ml/min, with a gradient elution consisting of acetonitrile and water containing 0.1% (v/v) formic acid at 30 °C and detected by electrospray ionization mass spectrometry in the positive multiple reaction monitoring (MRM) mode. The linearity was found to be within the concentration range of 0.5-5000 ng/ml with a lower limit of quantification of 0.5 ng/ml. The absolute oral bioavailability of hederasaponin B was 0.24 ± 0.49%. This indicated that the concentration-time course of the hederasaponin B existed a double-peak phenomenon. This method was further applied to the determination of hederasaponin B in rat plasma and showed good practicability, for the first time, after intragastric (25 mg/kg) and intravenous (2 mg/kg) administration in rats. [ABSTRACT FROM AUTHOR]

A rapid and sensitive high-performance liquid chromatographic (HPLC) method had been developed and validated for the analysis of three main ingredients in analgesic bacteriostatic gel. Hydroxy-α-sanshool (HAS), hydroxyl-β-sanshool (HBS) and hydroxy-γ-sanshool (HRS) were separated on a Thermo Hypersil GOLD C18 column (150 mm x 4.6 mm, 5 µm) with gradient elution system. The mobile phase consisted of acetonitrile-water (v/v) with a flow rate of 1.0 mL/min and UV detection was performed at 270 nm. The method was validated according to the Chinese Pharmacopeia (2015). Linearity (r > 0.9990) was observed over the concentration ranges of 3.24-58.32, 0.656-11.81 and 0.304-5.48 mg/L for HAS, HBS and HRS. The average recovery for HAS, HBS and HRS was 96.9%, 97.2% and 97.9%. The method was found simple and rapid, which can be suitable for the determination of the three main ingredients in analgesic bacteriostatic gel. [ABSTRACT FROM AUTHOR]