Selection and evaluation of quality markers (Q-markers) of vladimiriae radix extract for cholestatic liver injury based on spectrum-effect relationship, pharmacokinetics, and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: As a representative local medicinal herb produced in China, Vladimiriae Radix (VR) has been proven to exert hepatoprotective and choleretic effects, with particular therapeutic efficacy in cholestatic liver injury (CLI), as demonstrated by the VR extract (VRE). However, the quality markers (Q-markers) of VRE for the treatment of CLI remain unclear. AIM OF THE STUDY: A new strategy based on the core element of "efficacy" was proposed, using a combination of spectrum-effect relationship, pharmacokinetics, and molecular docking methods to select and confirm Q-markers of VRE. MATERIAL AND METHODS: First, the HPLC fingerprinting of 10 batches of VRE was studied, and the in vivo pharmacological index of anti-CLI in rats was determined. The spectrum-effect relationship was utilized as a screening method to identify the Q-markers of VRE. Secondly, Q-markers were used as VRE pharmacokinetic markers to measure their concentrations in normal and CLI rat plasma, and to analyze their disposition. Finally, molecular docking was utilized to predict the potential interaction between the identified Q-markers and crucial targets of CLI. RESULTS: The fingerprints of 10 batches of VRE was established. The in vivo pharmacological evaluation of rats showed that VRE had a significant therapeutic effect on CLI. The spectrum-effect correlation analysis showed that costunolide (COS) and dehydrocostus lactone (DEH) were the Q-markers of VRE anti-CLI. The pharmacokinetic results showed that AUC(0-t), Cmax, CLZ/F, and VZ/F of COS and DEH in CLI rats had significant differences (P < 0.01). They were effectively absorbed into the blood plasma of CLI rats, ensuring ideal bioavailability, and confirming their role as Q-markers. Molecular docking results showed that COS, DEH had good affinity with key targets (FXR, CAR, PXR, MAPK, TGR5, NRF2) for CLI treatment (Binding energy < -4.52 kcal mol-1), further verifying the correctness of Q-marker selection. CONCLUSIONS: In this study, through the combination of experimental and theoretical approaches from the aspects of pharmacodynamic expression, in vivo process rules, and interaction force prediction, the therapeutic effect of VRE and Q-markers (COS、DEH) were elucidated. Furthermore, a new idea based on the principle of "efficacy" was successfully proposed for screening and evaluating Q-markers.

Comparative transcriptome among Euscaphis konishii Hayata tissues and analysis of genes involved in flavonoid biosynthesis and accumulation.

BACHGROUND: Euscaphis konishii Hayata, a member of the Staphyleaceae Family, is a plant that has been widely used in Traditional Chinese Medicine and it has been the source for several types of flavonoids. To identify candidate genes involved in flavonoid biosynthesis and accumulation, we analyzed transcriptome data from three E. konishii tissues (leaf, branch and capsule) using Illumina Hiseq 2000 platform. RESULTS: A total of 91.7, 100.3 and 100.1million clean reads were acquired for the leaf, branch and capsule, respectively; and 85,342 unigenes with a mean length of 893.60 bp and N50 length of 1307 nt were assembled using Trinity program. BLASTx analysis allowed to annotate 40,218 unigenes using public protein databases, including NR, KOG/COG/eggNOG, Swiss-Prot, KEGG and GO. A total of 14,291 (16.75%) unigenes were assigned to 128 KEGG pathways, and 900 unigenes were annotated into 22 KEGG secondary metabolites, including flavonoid biosynthesis. The structure enzymes involved in flavonoid biosynthesis, such as phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate CoA ligase, shikimate O-hydroxycinnamoyltransferase, coumaroylquinate 3'-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase, flavonolsynthese, dihydroflavonol 4-reductase, anthocyanidinreductase, leucoanthocyanidin dioxygenase, leucoanthocyanidin reductase, were identified in the transcriptome data, 40 UDP-glycosyltransferase (UGT), 122 Cytochrome P450 (CYP) and 25 O-methyltransferase (OMT) unigenes were also found. A total of 295 unigenes involved in flavonoid transport and 220 transcription factors (97 MYB, 84 bHLH and 39 WD40) were identified. Furthermore, their expression patterns among different tissues were analyzed by DESeq, the differentially expressed genes may play important roles in tissues-specific synthesis, accumulation and modification of flavonoids. CONCLUSION: We present here the de novo transcriptome analysis of E. konishii and the identification of candidate genes involved in biosynthesis and accumulation of flavonoid. In general, these results are an important resource for further research on gene expression, genomic and functional genomics in E. konishii and other related species.

Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data.

BACKGROUND: Quantitative real-time reverse transcription-polymerase chain reaction has been widely used in gene expression analysis, however, to have reliable and accurate results, reference genes are necessary to normalize gene expression under different experimental conditions. Several reliable reference genes have been reported in plants of Traditional Chinese Medicine, but none have been identified for Euscaphis konishii Hayata. RESULTS: In this study, 12 candidate reference genes, including 3 common housekeeping genes and 9 novel genes based on E. konishii Hayata transcriptome data were selected and analyzed in different tissues (root, branch, leaf, capsule and seed), capsule and seed development stages. Expression stability was calculated using geNorm and NormFinder, the minimal number of reference genes required for accurate normalization was calculated by Vn/Vn + 1 using geNorm. EkEEF-5A-1 and EkADF2 were the two most stable reference genes for all samples, while EkGSTU1 and EkGAPDH were the most stable reference genes for tissue samples. For seed development stages, EkGAPDH and EkEEF-5A-1 were the most stable genes, whereas EkGSTU1 and EkGAPDH were identified as the two most stable genes in the capsule development stages. Two reference genes were sufficient to normalize gene expression across all sample sets. CONCLUSION: Results of this study revealed that suitable reference genes should be selected for different experimental samples, and not all the common reference genes are suitable for different tissue samples and/or experimental conditions. In this study, we present the first data of reference genes selection for E. konishii Hayata based on transcriptome data, our data will facilitate further studies in molecular biology and gene function on E. konishii Hayata and other closely related species.