Excretion characteristics of main compounds of Yigong San in urine, feces, and bile of rats.

Yigong San (YGS) is a traditional Chinese medicine formula used for pediatric anorexia, chronic atrophic gastritis, and irritable bowel syndrome. In this study, the excretion of eight main compounds, including liquiritin; isoliquiritin; hesperidin; ginsenosides Rb1, Re, and Rg1; and atractylenolides I and II, in rat urine, feces, and bile, was investigated by ultra-high performance liquid chromatography-tandem mass spectrometry. The results showed that the cumulative excretion rates of the compounds in rat urine, feces, and bile were 0.018-1.15%, 0.024-19.89%, and 0.0025-0.72%, respectively. Among the eight compounds detected, liquiritin was the richest in urine, and ginsenosides Re and Rg1 and atractylenolide I were mainly found in feces and bile. In summary, the main components of YGS are excreted via multiple approaches. Liquiritin is mainly through urine, whereas isoliquiritin; hesperidin; ginsenosides Rb1, Re, and Rg1; and atractylenolides I and II are mainly through feces. The excretion of these compounds in bile is usually positively correlated with that in feces. This study lays a foundation for further pharmacological research and application of YGS.

Dual catalytic potential of isoeugenol synthase in Asarum sieboldii Miq. (AsIGS): Unveiling isoeugenol preference in vitro and eugenol production in vivo, with insights into hydrogen bonding influence.

Asarum sieboldii Miq. is an important medicinal plant valued for its diverse health benefits in the pharmaceutical industry. In the present study, we isolated and characterized isoeugenol synthase from A. sieboldii (AsIGS), an essential enzyme involved in the biosynthesis of volatile phenylpropenes. We hoped to elucidate the secondary metabolic network of eugenol in A. sieboldii plants, which constructed the prerequisite for quality improvement of the well-known TCM Asari Radix et Rhizoma. Bioinformatics analysis revealed high similarity between the DNA sequences of AsIGS and isoeugenol synthase genes from other plants, and that the association of the candidate protein AsIGS with the PIP reductase family. Moreover, the AsIGS protein displayed a molecular weight of about 34.96 kDa, with a theoretical isoelectric point of 6.01 and an average hydrophobicity of -0.092, indicating the protein's partial acidity, stability, and hydrophilic nature. Phylogenetic analysis showed that AsIGS had a close relationship with isoeugenol synthases and fewer eugenol synthases found in other species. Alphafold2 predicted the structure of the AsIGS protein, and CB-Dock2 predicted the binding sites of the ASIGS-NADPH-coniferyl acetate ternary complex. In vitro enzymatic assay results demonstrated that the optimal temperature of the AsIGS-involved catalysis for coniferyl acetate was 30 °C, and several kinetics parameters were Km (12.21 mM), Vmax (27.9 U/mg), kcat (76.26 s-1), and kcat/Km (6.49 s-1·mM-1). Furthermore, it was also determined that the AsIGS protein had varying performance at different pH levels. While the candidate protein converted coniferyl acetate into both isoeugenol and eugenol at pH 5.5, it just catalyzed the production of isoeugenol at pH 6.5. However, isoeugenol has never been detected in A. sieboldii. Altering AsIGS expression in transgenic plants impacted only eugenol contents. Compared with wild type, overexpression of AsIGS increased eugenol content by 23.3 %, while RNAi-induced down-regulation of AsIGS decreased it by 25.3 %. Taken together, these results confirmed that the AsIGS gene was involved in the biosynthesis of eugenol in A. sieboldii with a dual catalytic potential.

Characterization and protein engineering of a novel UDP-glycosyltransferase involved in pseudoginsenoside Rt5 biosynthesis from Panax japonicus.

As one of rare high-value ocotillol (OCT)-type ginsenosides, pseudoginsenoside Rt5 has been identified with significant pharmacological activities. UDP-glycosyltransferases (UGTs) play pivotal roles in catalyzing the transfer of a glycosyl moiety from a donor to an acceptor. In this study, the novel UGT, PjUGT10, was screened from the transcriptome database of Panax japonicus and identified with the enzymatic activity of transferring a glucosyl group on OCT to produce Rt5. The catalytic efficiency of PjUGT10 was further enhanced by employing site-directed mutation. Notably, the variant M7 exhibited a remarkable 6.16 × 103-fold increase in kcat/Km towards 20S,24R-ocotillol and a significant 2.02 × 103-fold increase to UDP-glucose, respectively. Moreover, molecular dynamics simulations illustrated a reduced distance between 20S,24R-ocotillol and the catalytic residue His15 or UDP-glucose, favoring conformation interactions between the enzyme and substrates. Subsequently, Rt5 was synthesized in an engineered Escherichia coli strain M7 coupled with a UDP-glucose synthetic system. This study not only shed light on the protein engineering that can enhance the catalytic activity of PjUGT10, but also established a whole-cell approach for the production of Rt5.

Shengmai San formula alleviates high-fat diet-induced obesity in mice through gut microbiota-derived bile acid promotion of M2 macrophage polarization and thermogenesis.

BACKGROUND: Shengmai San Formula (SMS) is a traditional Chinese medicine (TCM) that has been used to treat wasting-thirst regarded as diabetes mellitus, which occurs disproportionately in obese patients. Therefore, we investigated whether SMS could be used to treat obesity, and explored possible mechanisms by which it might improve glucose and fat metabolism. METHODS: To investigate the effects of SMS on a high-fat diet (HFD)-induced obesity (DIO) model, we studied glucose metabolism via glucose tolerance testing (GTT) and insulin tolerance testing (ITT). Browning of white adipose tissue (WAT) was evaluated using H&E staining, along with browning-related gene and protein expression. Changes in bile acid (BA) levels in serum, liver, ileum, and inguinal white adipose tissue were detected by Ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). In addition, antimicrobial mixture (ABX) and fecal microbial transplantation (FMT) experiments were used to verify the role of gut flora in the effects produced by SMS on HFD-induced obesity model. RESULTS: SMS ameliorated diet-induced dyslipidemia in a dose-dependent manner and reduced glucose intolerance and insulin resistance in DIO mice, helping to restore energy metabolism homeostasis. SMS significantly altered the structure of intestinal microbiome composition, decreasing the abundance of Lactobacillus carrying bile salt hydrolase (BSH) enzymes and thereby increasing the level of conjugated BAs in the blood, ileum, and iWAT. Increased TCA content promoted the secretion of Slit3 from M2 macrophages in iWAT, which activates the protein kinase A/calmodulin-dependent protein kinase II (PKA/CaMKII) signaling pathway in sympathetic neurons via the roundabouts receptor 1(ROBO1). This pathway promotes the synthesis and release of norepinephrine (NE), inducing cyclic adenosine monophosphate (cAMP) release in adipose tissue that activates the cyclic adenosine monophosphate/protein kinase A/phosphorylated hormone-sensitive lipase (cAMP/PKA/pHSL) pathway and enhances WAT browning. ABX treatment eliminated SMS effects on glucose and lipid metabolism in DIO mice, whereas glucose and lipid metabolism in obese mice improved following SMS-FMT and increased the level of serum bile acids. CONCLUSION: SMS affects intestinal flora and bile acid composition in vivo and increased TCA promotes M2 macrophage polarization and Slit3 release in adipose tissue. This induces NE release and increases WAT browning in obese mice, which may be a mechanism by which SMS could be used to treat obesity.

Coupling the Isopentenol Utilization Pathway and Prenyltransferase for the Biosynthesis of Licoflavanone in Recombinant Escherichia coli.

Prenylflavonoids are promising candidates for food additives and functional foods due to their diverse biological activities and potential health benefits. However, natural prenylflavonoids are generally present in low abundance and are limited to specific plant species. Here, we report the biosynthesis of licoflavanone from naringenin and prenol by recombinant Escherichia coli. By investigating the activities of seven different sources of prenyltransferases overexpressed in E. coli toward various flavonoid substrates, the prenyltransferase AnaPT exhibits substrate preference when naringenin serves as the prenyl acceptor. Furthermore, licoflavanone production was successfully achieved by coupling the isopentenol utilization pathway and AnaPT in recombinant E. coli. In addition, the effects of fermentation temperatures, induction temperatures, naringenin concentrations, and substrate feeding strategies were investigated on the biosynthesis of licoflavanone in recombinant E. coli. Consequently, the recombinant E. coli strain capable of improved dimethylallyl diphosphate (DMAPP) supply and suitable for prenylflavonoid biosynthesis increased licoflavanone titers to 142.1 mg/L in a shake flask and to 537.8 mg/L in a 1.3 L fermentor, which is the highest yield for any prenylflavonoids reported to date. These strategies proposed in this study provide a reference for initiating the production of high-value prenylflavonoids.

Identification of mitophagy and ferroptosis-related hub genes associated with intracerebral haemorrhage through bioinformatics analysis.

BACKGROUND: Mitophagy and ferroptosis occur in intracerebral haemorrhage (ICH) but our understanding of mitophagy and ferroptosis-related genes remains incomplete. AIM: This study aims to identify shared ICH genes for both processes. METHODS: ICH differentially expressed mitophagy and ferroptosis-related genes (DEMFRGs) were sourced from the GEO database and literature. Enrichment analysis elucidated functions. Hub genes were selected via STRING, MCODE, and MCC algorithms in Cytoscape. miRNAs targeting hubs were predicted using miRWalk 3.0, forming a miRNA-hub gene network. Immune microenvironment variances were assessed with MCP and TIMER. Potential small molecules for ICH were forecasted via CMap database. RESULTS: 64 DEMFRGs and ten hub genes potentially involved in various processes like ferroptosis, TNF signalling pathway, MAPK signalling pathway, and NF-kappa B signalling pathway were discovered. Several miRNAs were identified as shared targets of hub genes. The ICH group showed increased infiltration of monocytic lineage and myeloid dendritic cells compared to the Healthy group. Ten potential small molecule drugs (e.g. Zebularine, TWS-119, CG-930) were predicted via CMap. CONCLUSION: Several shared genes between mitophagy and ferroptosis potentially drive ICH progression via TNF, MAPK, and NF-kappa B pathways. These results offer valuable insights for further exploring the connection between mitophagy, ferroptosis, and ICH.

Pharmacokinetics and tissue distribution of Yigong San in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM), Yigong San (YGS) is mainly used to treat dyspepsia caused by deficiency of spleen and stomach qi. Although the chemical composition and bioactivity of YGS has been well studied, the main in vivo compounds and their distribution in tissues still need to be made clearer. AIM OF THE STUDY: To elucidate the pharmacokinetic profiles and tissue distribution of eight main compounds of YGS in rats, and provide a reference for clinical application and new drug development. MATERIALS AND METHODS: UPLC-Q-Exactive-Orbitrap-MS was used to qualitatively characterize the parent compounds and their metabolites in the plasma of rats after oral administration of YGS. A sensitive, reliable, and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method using UPLC-AB Sciex QTRAP 5500 MS was established to quantitatively determine eight main compounds of YGS in rat plasma and tissues, including liquiritin, isoliquiritin, hesperidin, ginsenosides Rb1, Re and Rg1, atractylenolides I and II. RESULTS: The mean area under the concentration-time curve (AUC) values of ginsenoside Rb1, hesperidin, and liquiritin at low, medium, and high doses were greater than 150 ng h/mL. The elimination half-life (t1/2) values of ginsenoside Rb1, atractylenolides I and II (low and medium doses) were longer than 10 h. Peak time (Tmax) values of all compounds were shorter than 10 h. Except for atractylenolides, the maximum concentration (Cmax) values of the compounds were greater than 10 ng/mL. The eight compounds were detected in the heart, brain, liver, spleen and kidney at 0.25 h after oral administration. Liquiritin and isoliquiritin had higher exposure in the liver and heart. Hesperidin and ginsenosides Rb1, Re, and Rg1 are mainly distributed in the spleen and kidney. Atractylenolides I and II are mainly distributed in spleen, liver and kidney. CONCLUSIONS: All main compounds of YGS, i.e., liquiritin, isoliquiritin, hesperidin, ginsenosides Rb1, Re, and Rg1, and atractylenolides I and II are absorbed into plasma and widely distributed in various tissues. Among them, hesperidin, ginsenoside Rb1, and atractylenolide I are main in vivo compounds. They are mainly distributed in spleen, liver and kidney. The results of this study provide a basis for further in-depth development and application of YGS.

Novel glycosidase from Paenibacillus lactis 154 hydrolyzing the 28-O-ß-D-glucopyranosyl ester bond of oleanane-type saponins.

Oleanane-type ginsenosides are a class of compounds with remarkable pharmacological activities. However, the lack of effective preparation methods for specific rare ginsenosides has hindered the exploration of their pharmacological properties. In this study, a novel glycoside hydrolase PlGH3 was cloned from Paenibacillus lactis 154 and heterologous expressed in Escherichia coli. Sequence analysis revealed that PlGH3 consists of 749 amino acids with a molecular weight of 89.5 kDa, exhibiting the characteristic features of the glycoside hydrolase 3 family. The enzymatic characterization results of PlGH3 showed that the optimal reaction pH and temperature was 8 and 50 °C by using p-nitrophenyl-ß-D-glucopyranoside as a substrate, respectively. The Km and kcat values towards ginsenoside Ro were 79.59 ± 3.42 µM and 18.52 s-1, respectively. PlGH3 exhibits a highly specific activity on hydrolyzing the 28-O-ß-D-glucopyranosyl ester bond of oleanane-type saponins. The mechanism of hydrolysis specificity was then presumably elucidated through molecular docking. Eventually, four kinds of rare oleanane-type ginsenosides (calenduloside E, pseudoginsenoside RP1, zingibroside R1, and tarasaponin VI) were successfully prepared by biotransforming total saponins extracted from Panax japonicus. This study contributes to understanding the mechanism of enzymatic hydrolysis of the GH3 family and provides a practical route for the preparation of rare oleanane-type ginsenosides through biotransformation. KEY POINTS: • The glucose at C-28 in oleanane-type saponins can be directionally hydrolyzed. • Mechanisms to interpret PlGH3 substrate specificity by molecular docking. • Case of preparation of low-sugar alternative saponins by directed hydrolysis.

Combinatorial metabolic engineering of Bacillus subtilis enables the efficient biosynthesis of isoquercitrin from quercetin.

BACKGROUND: Isoquercitrin (quercetin-3-O-ß-D-glucopyranoside) has exhibited promising therapeutic potentials as cardioprotective, anti-diabetic, anti-cancer, and anti-viral agents. However, its structural complexity and limited natural abundance make both bulk chemical synthesis and extraction from medical plants difficult. Microbial biotransformation through heterologous expression of glycosyltransferases offers a safe and sustainable route for its production. Despite several attempts reported in microbial hosts, the current production levels of isoquercitrin still lag behind industrial standards. RESULTS: Herein, the heterologous expression of glycosyltransferase UGT78D2 gene in Bacillus subtilis 168 and reconstruction of UDP-glucose (UDP-Glc) synthesis pathway led to the synthesis of isoquercitrin from quercetin with titers of 0.37 g/L and 0.42 g/L, respectively. Subsequently, the quercetin catabolism blocked by disruption of a quercetin dioxygenase, three ring-cleavage dioxygenases, and seven oxidoreductases increased the isoquercitrin titer to 1.64 g/L. And the hydrolysis of isoquercitrin was eliminated by three ß-glucosidase genes disruption, thereby affording 3.58 g/L isoquercitrin. Furthermore, UDP-Glc pool boosted by pgi (encoding glucose-6-phosphate isomerase) disruption increased the isoquercitrin titer to 10.6 g/L with the yield on quercetin of 72% and to 35.6 g/L with the yield on quercetin of 77.2% in a 1.3-L fermentor. CONCLUSION: The engineered B. subtilis strain developed here holds great potential for initiating the sustainable and large-scale industrial production of isoquercitrin. The strategies proposed in this study provides a reference to improve the production of other flavonoid glycosides by engineered B. subtilis cell factories.

Determining the role of innovative teaching practices, sustainable learning, and the adoption of e-learning tools in leveraging academic motivation for students' mental well-being.

BACKGROUND: The COVID-19 pandemic has brought about significant changes in the educational landscape, with a significant shift towards e-learning and remote teaching practices. As such, it has become increasingly important to understand the role of innovative teaching practices, sustainable learning, and the adoption of e-learning tools in leveraging academic motivation for students' mental well-being. PURPOSE: The study aims to determine whether academic motivation can helpful for mental wellbeing of students directly and through the adoption of e-learning tools, and sustainable learning considering the role of innovative teaching. METHODS: Target population of this research were the students of Chinese universities. Data was collected from 308 students and was analyzed by using Mplus software. RESULTS: Students expressed higher motivation, quality education and good mental health. Additionally, it was discovered that academic motivation helped the students to develop good academic record and mental health. CONCLUSION: The research's conclusions can help the policy makers creating successful educational initiatives and programs that promote students' overall growth. These results can also guide the university administration and teachers to adopt effective policies and practices for creating academic motivation in order to construct a healthy environment not just for better academic results but also for the well-being of students. Additionally, this research draws attention of future researchers to explore mechanisms that can drive students' academic and psychological outcomes.

A bacteriophage against Citrobacter braakii and its synergistic effect with antibiotics.

A bacteriophage BD49 specific for Citrobacter braakii was screened out and purified by double-layer plate method. It consists of a polyhedral head of 93.1 ± 1.2 nm long and 72.9 ± 4.2 nm wide, tail fibers, collar, sheath and baseplate. The bacteriophage was identified by morphology observed with transmission electron microscope (TEM), whole genome sequencing carried out by Illumina next generation sequencing (NGS) technique, and gene annotation based on Clusters of Orthologous Groups of proteins (COG) database. It was identified primarily as a member of Caudovirales by morphology and further determined as Caudovirales, Myoviridae, and Citrobacter bacteriophage by alignment of its whole genome sequence with the NCBI database and establishment of phylogenetic tree. The bacteriophage showed good environmental suitability with optimal multiplicity of infection (MOI) of 0.01, proliferation time of 80 min, optimum living temperature of 30-40 °C, and living pH of 5-10. In addition, it exhibited synergistic effect with ciprofloxacin against C. braakii in antibacterial tests.

Lusianthridin ameliorates high fat diet-induced metabolic dysfunction-associated fatty liver disease via activation of FXR signaling pathway.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a common chronic liver disease, but there are few specific medications for it. Lusianthridin, a major phenanthrene component that originates from Dendrobium Sonia, has various in vitro biological functions. In this study, we aimed to evaluate the therapeutic effects of lusianthridin on high-fat diet (HFD)-induced MAFLD as well as to examine the mechanism of its effects. We fed male mice high-fat-diet for 12 weeks to induce MAFLD and then continued to feed them, either with or without lusianthridin, for another six weeks. We found that lusianthridin decreased serum triacylglycerol, hepatic triacylglycerol, and serum low density lipoprotein cholesterol. It also reduced hepatic lipid accumulation based on the results of morphology analysis. Besides, it improved hepatic inflammation as well, including a decrease in serum alanine aminotransferase and a reduction in macrophage and neutrophil infiltration. Mechanistically, surface plasmon resonance, cell thermal shift assay and dual-luciferase report system results suggested that lusianthridin combined with farnesoid X receptor (FXR) ligand binding region and activated its transcriptional activity. Lusianthridin also decreased de no lipogenesis though inhibiting Srebp1c and downstream Scd-1, Lpin1 and Dgat2 expression in a FXR-dependent manner in oleic acid treated L02 cells. Correspondingly, lusianthridin inhibited Srebp1c and downstream lipogenesis in MAFLD liver tissues of mice at both of genetic and protein levels. Finally, the protective effects of lusianthridin on hepatic steaotosis were abolished in Fxr-/- mice. Taken together, our results suggested that lusianthridin attenuated high-fat-diet induced MAFLD via activation the FXR signaling pathway.

Diversity and Isolation of Endophytic Fungi in Panax japonicus and Biotransformation Activity on Saponins.

OBJECTIVE: This study reports the diversity and community structure differences of the endophytic fungi of Panax japonicus of different ages to obtain novel endophytic fungi with glycoside hydrolytic activity for rare saponins production. METHODS: This study used the high-throughput sequencing method to analyze the diversity and community structure of endophytic fungi of P. japonicus. The endophytic fungi were processed by traditional isolation, culture, conservation, and ITS rDNA sequence analyses. Then the total saponins of P. japonicus were used as the substrate to evaluate the glycoside hydrolytic activity. RESULTS: The composition analysis of the community structure showed that the abundance, evenness, and diversity of endophytic fungi of nine-year-old P. japonicus were the best among all samples. A total of 210 endophytic fungi were isolated from P. japonicus samples and further annotated by sequencing the internal transcribed spacer. Then the biotransformation activity of obtained strains was further examined on total saponins of P. japonicus (TSPJ), with a strain identified as Fusarium equiseti (No.30) from 7-year-old P. japonicus showing significant glycoside hydrolytic activity on TSPJ, including ginsenoside Ro→zinglbroside R1, pseudoginsenoside RT1→pseudoginsenoside RP1, chikusetsusaponin IV→tarasaponin VI and chikusetsusaponin IVa →calenduloside E. CONCLUSION: These results reveal the diversity and community structure differences of the endophytic fungi of P. japonicus with different ages and establish a resource library of endophytic fungi of P. japonicus. More importantly, we identified a valuable endophytic fungus with glycoside hydrolytic activity and provided a promising convenient microbial transformation approach to produce minor deglycosylated ginsenosides.

Red ginseng extracts ameliorate high-fat diet-induced obesity and insulin resistance by activating the intestinal TGR5-mediated bile acids signaling pathway.

BACKGROUND: Obesity has emerged as a worldwide metabolic disease, given its rapid growth in global prevalence. Red ginseng extracts (RGS), one of the traditional processed products of ginseng, show the potential to improve the metabolic phenotype of obesity. However, the RGS mechanism for regulating obesity and late insulin resistance remains to be clarified. PURPOSE: This study aimed to emphasize the potential use of RGS in treatment of obesity and insulin resistance (IR) and explore the underlying mechanism affecting glucose and lipid metabolism improvements. METHODS: The role of RGS was evaluated in a high-fat diet (HFD) rodent model. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed to characterize the glucose metabolism level. The expression of lipolysis proteins and uncoupling protein-1 (UCP-1) were investigated by western blot. Glucagon-like peptide-1 (GLP-1) and apical sodium-dependent bile acid transporter (ASBT) protein expression in the intestine were determined via immunofluorescence. UPLC-Q-TOF-MS were used to detect the alterations in bile acids (BAs) levels in serum, ileum, and inguinal white adipose tissue (iWAT). In addition, intestine-specific Tgr5 knockout mice were employed to verify the efficacy of RGS in improving obesity. RESULTS: RGS treatment alleviated dietary-induced dyslipidemia and IR in obese mice in a dose-dependent manner and improved glucose and insulin tolerance, and energy expenditure. RGS treatment significantly reduced lipid deposition and induced GLP-1 secretion in the intestine of wild-type mice but not in Tgr5ΔIN obese mice. Furthermore, RGS intervention increased BA levels in serum, ileum, and iWAT. The increase of circulating BAs in mice was related to the activation of ileal TGR5 and the promotion of ASBT translocation to the plasma membrane, thus affecting BA transport. Next, the increased level of circulating BAs entered the periphery, which might facilitate lipolysis and energy consumption by activating TGR5 in iWAT. CONCLUSION: Our results demonstrated that RGS significantly alleviated HFD-induced obesity and insulin resistance in mice. RGS intervention improved glucose metabolism, promoted lipolysis, and energy metabolism by activating TGR5 in the intestine. In addition, we found that activating intestinal TGR5 facilitated the localization of ASBT to the plasma membrane, which ultimately promoted the transport of BAs to regulate metabolic phenotype.

Screening quality markers (Q-markers) of Xiaoer Chaige Tuire Oral Liquid by in vitro sequential metabolism and in vivo biopharmaceutical analysis.

BACKGROUND: Xiaoer Chaige Tuire Oral Liquid (XCT) is a preparation composed of 7 traditional Chinese medicines including Bupleuri Radix, Puerariae Lobatae Radix, Scutellariae Radix, Gypsum Fibrosum, Artemisiae Annuae Herba, Paeoniae Radix Alba and Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle in proportion. According to traditional Chinese medicine theory, it has the function of dispelling wind evil and relieving exterior syndrome, clearing summer heat and dampness, and reducing internal heat. So, it is indicated for pediatric upper respiratory tract infection caused by exogenous wind-heat. Modern pharmacological studies have indicated that XCT has a variety of activities such as anti-inflammation and antivirus. PURPOSE: To screen potential quality markers (Q-markers) of XCT by tracking in vivo bioactive compounds concomitantly using in vitro sequential metabolism and in vivo biopharmaceutical analysis. METHODS: In vitro metabolic models including artificial gastric juice, intestinal juice, intestinal microbiota, Caco-2 cell monolayer and liver S9 were employed to simulate metabolism of main compounds of XCT in the body. High performance liquid chromatography with diode-array detection (HPLC-DAD) was used to quantitatively determine main components of XCT preparation and its sequential metabolism samples. Ultra performance liquid chromatography with QExactive Orbitrap tandem mass spectrometry (UPLC-QExactive-HF-x-Orbitrap-MS) was used to qualitatively determine in vivo components of XCT preparation in rat plasma and metabolites obtained with liver S9 fraction of rats. RESULTS: Twenty-five compounds were identified from the preparation of XCT. Sequential in vitro metabolism studies indicated that most of these compounds except baicalin and baicalein were stable in artificial gastric juice, albiflorin, glycyrrhizic acid, gallic acid and baicalein were unstable in artificial intestinal juice, daidzin, liquiritin and genistin were hydrolyzed into their aglycones daidzein, liquiritigenin and genistein by intestinal microbiota, and 7 compounds thereout including benzoic acid, puerarin, 3'-methoxypuerarin, paeoniflorin, scopoletin, daidzein and liquiritigenin were shown to be well absorbed with Caco-2 cell monolayer model. These 7 compounds were demonstrated to be metabolized via hydroxylation and glycosylation by liver S9 system. Ten components of XCT preparation including puerarin, baicalin, wogonoside, benzoic acid, daidzein, baicalein, wogonin, oroxylin A, isoscopoletin and isoliquiritigenin were identified from rat plasma by in vivo biopharmaceutical analysis. Most of the compounds screened with both in vitro and in vivo metabolic studies were shown to be active against inflammation and influenza virus. CONCLUSIONS: A screening strategy for potential quality markers (Q-markers) of XCT preparation based on tracking in vivo bioactive compounds using the combination of in vitro sequential metabolism and in vivo biopharmaceutical analysis was established. With this strategy, a total of 12 compounds including puerarin, daidzein, benzoic acid, baicalin, baicalein, wogonoside, wogonin, oroxylin A, 3'-methoxypuerarin, paeoniflorin, scopoletin and liquiritigenin were screened to be potential Q-markers of XCT, which provides a material basis for quality control and development of XCT.

MicroRNA-142-5p promotes the proliferation and metastasis of nasopharyngeal carcinoma.

Growing pieces of evidence reported abnormal expression of microRNA in various cancer. Our research aimed to ascertain the miR-142-5p expression and its potential function in the growth and metastasis of human nasopharyngeal carcinoma (NPC). In human NPC tissues and cell lines, miR-142-5p expression was quantified via the real-time qPCR assay. Functionally, the potential effect of miR-142-5p in human CNE-1 and SUNE-1 cells through MTT assay, colony formation assay, Transwell assay, and cell cycle assay. In addition, the potential target gene of miR-142-5p was determined by the dual-luciferase reporter assay. MiR-142-5p expression was remarkably elevated in human NPC tissues, CNE-1 and SUNE-1 cells. MiR-142-5p overexpression obviously enhanced the ability of cell proliferative and colony formation, and prevented G1 phase arrest in CNE-1 and SUNE-1 cells. Further, the migration number of NPC cells was increased compared to NP69 cells. BTG3 was identified as the direct target gene of miR-142-5p. Inhibition of BTG3 expression could reverse the cell proliferation by miR-142-5p-induced. Overall, miR-142-5p could strengthen the NPC cell's proliferation and migration by directly targeting BTG3. Hence, miR-142-5p may provide a new strategy and program for future clinical treatment of NPC.

Combined treatment with Prunella vulgaris and Radix bupleuri activated the Bax/Bcl-2 and Caspase-3 signal pathways in papillary thyroid carcinoma cells.

To explore the effect of Prunella vulgaris (PV) combined with Radix bupleuri (RB) on apoptosis of papillary thyroid carcinoma cells. Our study was divided into four groups: the control group, the PV group, the RB group, and the PV combined with the RB group. The viability of cells from different treatment groups was assessed by the CCK-8 assay. Cell migration and invasion were assessed by healing wounding and the transwell assay, respectively. Cell apoptosis rate and cell cycle arrest were detected by a flow cytometry assay. The protein expression of Bcl-2, Bax, Caspase-3, CyclinA1, CyclinB1, and CDK1 was detected using a western blot assay. Our results indicated that, compared with the control group, PV combined with RB group could significantly alter the cell morphology, inhibit cell migration and invasion, decrease the number of cells in the G0/G1 phase and increase the number of cells in the G2/M phase, and promote the cell apoptosis. Moreover, PV combined with RB treatment also obviously increased the expression of Bax/Bcl2 and caspase-3 proteins and decreased the expression of Cyclin A1, Cyclin B1, and CDK1 proteins. Overall, our results indicated that PV combined with RB could activate the Bax/Bcl-2 and Caspase-3 signal pathways to induce cell apoptosis in papillary thyroid carcinoma cells; this also provides a new way to treat thyroid cancer.

Interaction between Changan Granule and its main components in the plasma and CYP450 enzymes.

ETHNOPHARMACOLOGICAL RELEVANCE: Changan Granule (CAG) is a Chinese patent drug developed based on an empirical prescription in accordance with the formulation theory of Traditional Chinese Medicine. The prescription is composed of eight herbal drugs which have been traditionally used by Chinese people for a long history. It has effects of invigorating spleen and supplementing qi, as well as regulating liver and ceasing diarrhea, and is indicated for the treatment of irritable bowel syndrome (IBS). AIM OF THE STUDY: This study was aimed to investigate the interaction between CAG and its main components and cytochrome P450 (CYP450) enzymes so as to characterize the major metabolites and metabolic enzymes and evaluate the safety concerns to its clinical use. MATERIALS AND METHODS: Both in vivo and in vitro experiments using such as diarrhea-predominant IBS (IBS-D) rat model, HepG2 cells, and human liver microsomes (HLM) were carried out to investigate the interaction between CAG and its main components and CYP450 enzymes. Real-time quantitative PCR (qPCR), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and cocktail probes were employed to qualitatively or quantitatively measure the metabolites and metabolic enzymes. RESULTS: CAG inhibited the enzyme activities of CYP1A2, CYP2E1, CYP2D6, CYP2C9, and CYP3A4 and the mRNA expressions of CYP2E1, CYP2C9, CYP3A4, and CYP2D6 in vitro. CAG down-regulated the increased expression of CYP1A2 and up-regulated the decreased expression of CYP3A1 in vivo. Twenty-two metabolites were characterized from the main components of CAG after incubation with HLM in vitro. CYP2D6, CYP2E1, CYP3A4 and CYP2C9 were identified as the characteristic metabolic enzymes. CONCLUSIONS: This study provides a reference for clinical application of CAG in safety. CAG and CYP450 enzymes are interacted. CAG is mainly metabolized by CYP2E1 and CYP2D6. The expression of CYP2E1 and CYP2D6 are more susceptible to be influenced by CAG in comparison with that of CYP3A4, CYP2C9 and CYP1A2. It implies the potential risk of interaction when CAG is taken together with the drugs metabolized by CYP2E1 and CYP2D6.

Full-length transcriptome, proteomics and metabolite analysis reveal candidate genes involved triterpenoid saponin biosynthesis in Dipsacus asperoides.

Dipsacus asperoides is a traditional medicinal herb widely used in inflammation and fracture in Asia. Triterpenoid saponins from D. asperoides are the main composition with pharmacological activity. However, the biosynthesis pathway of triterpenoid saponins has not been completely resolved in D. asperoides. Here, the types and contents of triterpenoid saponins were discovered with different distributions in five tissues (root, leaf, flower, stem, and fibrous root tissue) from D. asperoides by UPLC-Q-TOF-MS analysis. The discrepancy between five tissues in D. asperoides at the transcriptional level was studied by combining single-molecule real-time sequencing and next- generation sequencing. Meanwhile, key genes involved in the biosynthesis of saponin were further verified by proteomics. In MEP and MVA pathways, 48 differentially expressed genes were identified through co-expression analysis of transcriptome and saponin contents, including two isopentenyl pyrophosphate isomerase and two 2,3-oxidosqualene ß-amyrin cyclase, etc. In the analysis of WGCNA, 6 cytochrome P450s and 24 UDP- glycosyltransferases related to the biosynthesis of triterpenoid saponins were discovered with high transcriptome expression. This study will provide profound insights to demonstrate essential genes in the biosynthesis pathway of saponins in D. asperoides and support for the biosynthetic of natural active ingredients in the future.