Insufficient catalytic efficiency of flavonoid 6-hydroxylases in the fermentative production of scutellarin leads to the formation of at least about 18% of by-products. Here, the catalytic mechanisms of two flavonoid 6-hydroxylases, CYP82D4 and CYP706X, were investigated by molecular dynamics simulations and quantum chemical calculations. Our results show that CYP82D4 and CYP706X have almost identical energy barriers at the rate-determining step and thus similar reaction rates, while the relatively low substrate binding energy of CYP82D4 may facilitate product release, which is directly responsible for its higher catalytic efficiency. Based on the study of substrate entry and release processes, the catalytic efficiency of the L540A mutation of CYP82D4 increased by 1.37-fold, demonstrating the feasibility of theoretical calculations-guided engineering of flavonoid 6-hydroxylase. Overall, this study reveals the catalytic mechanism of flavonoid 6-hydroxylases, which may facilitate the modification and optimization of flavonoid 6-hydroxylases for efficient fermentative production of scutellarin.
Cytochrome P-450 Enzyme System/metabolism* ; Apigenin ; Glucuronates

Dead heart is an important trait of pith-decayed Scutellariae Radix. The purpose of this study was to clarify the scientific connotation of the dead heart using multi-omics. Metabolomics and transcriptomics combined with multivariate statistical analysis such as principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA) were used to systematically compare the differences in chemical composition and gene expression among phloem, outer xylem and near-dead xylem of pith-decayed Scutella-riae Radix. The results revealed significant differences in the contents of flavonoid glycosides and aglycones among the three parts. Compared with phloem and outer xylem, near-dead xylem had markedly lowered content of flavonoid glycosides(including baicalin, norwogonin-7-O-β-D-glucuronide, oroxylin A-7-O-β-D-glucuronide, and wogonoside) while markedly increased content of aglycones(including 3,5,7,2',6'-pentahydroxy dihydroflavone, baicalin, wogonin, and oroxylin A). The differentially expressed genes were mainly concentrated in KEGG pathways such as phenylpropanoid metabolism, flavonoid biosynthesis, ABC transporter, and plant MAPK signal transduction pathway. This study systematically elucidated the material basis of the dead heart of pith-decayed Scutellariae Radix with multiple growing years. Specifically, the content of flavonoid aglycones was significantly increased in the near-dead xylem, and the gene expression of metabolic pathways such as flavonoid glycoside hydrolysis, interxylary cork development and programmed apoptosis was significantly up-regulated. This study provided a theoretical basis for guiding the high-quality production of pith-decayed Scutellariae Radix.
Drugs, Chinese Herbal/chemistry* ; Scutellaria baicalensis/chemistry* ; Glucuronides ; Multiomics ; Flavonoids/chemistry*

Drinking culture has high significance in both China and the world, whether in the entertainment sector or in social occasions; according to the World Health Organization's 2018 Global Alcohol and Health Report, about 3 million people died from excessive drinking in 2016, accounting for 5.3% of the total global deaths that year. Oxidative stress and inflammation are the most common pathological phenomena caused by alcohol abuse (Snyder et al., 2017). Scutellarin, a kind of flavonoid, is one of the main active ingredients extracted from breviscapine. It exerts anti-inflammatory, antioxidant, and vasodilation effects, and has been used to treat cardiovascular diseases and alcoholic liver injury. Although scutellarin can effectively alleviate multi-target organ injury induced by different forms of stimulation, its protective effect on alcoholic brain injury has not been well-defined. Therefore, the present study established an acute alcohol mice brain injury model to explore the effect of scutellarin on acute alcoholic brain injury. The study was carried out based on the targets of oxidative stress and inflammation, which is of great significance for the targeted therapy of clinical alcohol diseases.
Animals ; Apigenin/therapeutic use* ; Brain Injuries/drug therapy* ; Glucuronates/therapeutic use* ; Humans ; Mice ; Oxidative Stress

Mucic acid is a hexaric acid that can be biosynthesized by oxidation of D-galacturonic acid, which is the main constituent of pectin. The structure and properties of mucic acid are similar to that of glucaric acid, and can be widely applied in the preparation of important platform compounds, polymers and macromolecular materials. Pectin is a cheap and abundant renewable biomass resource, thus developing a process enabling production of mucic acid from pectin would be of important economic value and environmental significance. This review summarized the structure and hydrolysis of pectin, the catabolism and regulation of D-galacturonic acid in microorganisms, and the strategy for mucic acid production based on engineering of corresponding pathways. The future application of mucic acid are prospected, and future directions for the preparation of mucic acid by biological method are also proposed.
Hexuronic Acids/metabolism* ; Pectins/metabolism* ; Sugar Acids/metabolism*

According to the taste analysis of Pudilan Xiaoyan Oral Liquid, the unpleasant taste of the oral liquid is mainly caused by the inherent taste of Chinese medicine and the taste introduced in the preparation process, which leads to its unpopularity among children. Therefore, aiming at the special children patient group, Xiaoer Pudilan Xiaoyan Syrup was developed via technology optimization and dosage form improvement to improve the unpleasant taste and enhance the medication compliance among children. Based on the material properties of Pudilan Xiaoyan Oral Liquid and Xiaoer Pudilan Xiaoyan Syrup extracts, the authors compared the properties(pH, density, turbidity, viscosity, chromaticity, particle size), taste, content of five quality markers and in vivo pharmacokinetic characteristics of these two preparations, to evaluate the suitability of Xiaoer Pudilan Xiaoyan Syrup. The results showed that compared with those of Pudilan Xiaoyan Oral Liquid, the pH, density, turbidity, viscosity and chromaticity of Xiaoer Pudilan Xiaoyan Syrup were significantly changed, and the unpleasant taste was reduced by 26%; the transfer rate of the main active ingredients chicoric acid was increased, while the transfer rate of baicalin had small difference from that of the oral liquid. In addition, pharmacokinetics revealed that the total absorption amount of baicalin in vivo was higher, and the time to peak T_(max) of baicalin and oroxindin in the syrup and the mean residence time MRT_(last )of corynoline in vivo were significantly prolonged. The absorption degree of Xiaoer Pudilan Xiaoyan Syrup and Pudilan Xiaoyan Oral Liquid in the body was the same: baicalin>oroxindin>corynoline. The new dosage form process was simpler than that of the original dosage form, safe, environmentally friendly, reasonable and feasible, meeting the mass production demand. This provided a basis for the reasonable and scientific optimization of Xiaoer Pudilan Xiaoyan Syrup, and also laid a foundation for its further safe and rational use, so as to expand the clinical application in children.
Child ; Humans ; Drugs, Chinese Herbal ; Glucuronates

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 μL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 μL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 μL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 μL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 μL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 μL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 μL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 μL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 μL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 μL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 μL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 μL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 μL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.
Animals ; Benzofurans ; Coumarins ; Dogs ; Glucuronides ; Glucuronosyltransferase/metabolism* ; Kinetics ; Mice ; Microsomes, Liver/metabolism* ; Phenotype ; Rats ; Species Specificity ; Swine ; Swine, Miniature/metabolism*

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.
Animals ; Carbohydrate Epimerases ; biosynthesis ; chemistry ; genetics ; Escherichia coli ; Gene Expression ; Heparin ; metabolism ; Heparitin Sulfate ; metabolism ; Iduronic Acid ; metabolism ; Zebrafish Proteins ; biosynthesis ; chemistry ; genetics

To define the composition of relevant substances in Breviscapine for Injection, in order to improve the quality control of impurity, and ensure the clinical safety. The analysis and structural identification of relevant substances in different specifications and batches of Breviscapine for Injection powders were carried out by HPLC and UPLC-QTOF-MS. Three primary relevant substances, namely 5,6,7,3',4'-pentahydroxyflavone-7-O-glucuronide(3), 3,5,6,7,4'-pentahydroxyflavone-3-O-glucuronide(4) and scutellarein(10), as well as three minor impurities, namely 6-hydroxyapigenin-6-O-glucosyl-7-O-glucuronide(1), methoxylscutellarin(6) and apigenin-7-O-glucuronide(7) were structurally identified by matching retention time, UV spectra, and mass spectra with authentic compounds and MS fragmentation rules. The main relevant substances(3) and(4) were separated and purified by semi-preparative HPLC, and their structures were further confirmed by NMR data. The study defined relevant substances of Breviscapine for Injection, and provided reference for improving the quality control level of single impurity in breviscapine preparation.
Apigenin/analysis* ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/standards* ; Flavonoids/chemistry* ; Glucuronides/analysis* ; Injections ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; Quality Control

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.
Animals ; Aspergillus niger ; Calibration ; Cellulase/analysis* ; Chemistry, Clinical/methods* ; Dextranase/analysis* ; Enterocolitis, Necrotizing/diagnosis* ; Equipment Design ; Flavonoids/analysis* ; Glucose/analysis* ; Glucuronic Acid/analysis* ; Glucuronidase/analysis* ; Glycoside Hydrolases/analysis* ; Hydrogen-Ion Concentration ; Linear Models ; Multienzyme Complexes/analysis* ; Plants, Medicinal ; Polygalacturonase/analysis* ; Rats ; Reproducibility of Results ; beta-Galactosidase/analysis* ; beta-Glucosidase/analysis*

In the present study, a gastric retention floating system for Brucea javanica oil, composed of alginate and carrageenan, was prepared using ionotropic gelation. Parameters for floatability, drug load, encapsulation efficiency, bead morphology, in vitro release, and in vivo gastric retention were evaluated. The optimized formulation via Box-Behnken design consisted of 1.7% alginate (W/V), 1.02% carrageenan (W/V), 1.4% CaCO (W/V), and a gelling bath of pH 0.8. The alginate-carrageenan-Brucea javanica oil beads had a porous structure and exhibited up to 24 h of in vitro floatability with a load capacity of 45%-55% and an encapsulation efficiency of 70%-80%. A 6-h sustained release was observed in vitro. The beads had a prolonged gastric retention (> 60% at 6 h) in fasted rats, compared to non-floating beads (15% at 6 h), as measured by gamma scintigraphy with single-photon emission tomography/computed tomography (SPET/CT). In conclusion, the alginate-carrageenan-Brucea javanica oil system showed enhanced oil encapsulation efficiency, excellent floating and gastric retention abilities, and a favorable release behavior.
Alginates ; chemistry ; Animals ; Biological Availability ; Brucea ; chemistry ; Carrageenan ; chemistry ; Delayed-Action Preparations ; administration & dosage ; chemistry ; pharmacokinetics ; Drug Carriers ; chemistry ; Drug Delivery Systems ; methods ; Drug Evaluation, Preclinical ; Gastric Mucosa ; metabolism ; Glucuronic Acid ; chemistry ; Hexuronic Acids ; chemistry ; Microspheres ; Plant Oils ; administration & dosage ; chemistry ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley