Flavonoid and chromone-rich extract from Euscaphis Konishii Hayata leaf attenuated alcoholic liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Euscaphis konishii Hayata is a traditional medicinal plant in China, and its leaves are usually used to make dishes for hepatic or gastrointestinal issues by Chinese She nationality. Pharmacological analysis showed that E. konishii leaves contain high levels of flavonoids and chromones with favorable anti-hepatoma effect. AIM OF THE STUDY: The extract from E. konishii leaves was detected to evaluate its chemical composition, and the alcoholic liver injury mice model was adopted to elucidate its hepatoprotective effects. MATERIALS AND METHODS: The total leaf extract from E. konishii was separated by polyamide column to get the flavonoid and chromone-rich extract (FCE). Single compounds from FCE was purified by gel and Sephadex LH-20 chromatography and analyzed by nuclear magnetic resonance (NMR). The chemical component of FCE was confirmed and quantified by HPLC-MS. The OH·, O2-, DPPH and ABTS + free radical assays were adopted to estimate the antioxidant activity of FCE in vitro. The alcohol-fed model mice were established to assess the hepatoprotective capacity of FCE in vivo, through biochemical determination, histopathological analysis, mitochondrial function measurement, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) detection and Western blot determination. RESULTS: 8 flavonoids and 2 chromones were recognized in the FCEextract by both NMR and HPLC-MS. FCE represented strong free radicals scavenging activity in vitro. With oral administration, FCE (50, 100 and 200 mg/kg) dose-dependently decreased the serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in alcohol-fed mice. FCE gradually reduced the malondialdehyde (MDA) content, increased the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the alcohol-treated liver tissues. FCE also alleviated the hepatic inflammation, inhibited the hepatocyte apoptosis and lessened the alcohol-induced histological alteration and lipid accumulation in the liver tissues. FCE administration inhibited the overexpression of endoplasmic reticulum (ER) chaperones signaling and unfolded protein response (UPR) pathways to defense the ER-induced apoptosis. Pretreatment with FCE also restored the mitochondrial membrane potentials andadenosine triphosphate (ATP) levels, which in turn suppressed the Cytochrome C release and mitochondria-induced apoptosis. CONCLUSIONS: FCE conferred great protection against alcoholic liver injury, which might be associated with its viability through suppressing reactive oxygen species (ROS) stress and hepatocyte apoptosis.

The Euscaphis japonica genome and the evolution of malvids.

Malvids is one of the largest clades of rosids, includes 58 families and exhibits remarkable morphological and ecological diversity. Here, we report a high-quality chromosome-level genome assembly for Euscaphis japonica, an early-diverging species within malvids. Genome-based phylogenetic analysis suggests that the unstable phylogenetic position of E. japonica may result from incomplete lineage sorting and hybridization event during the diversification of the ancestral population of malvids. Euscaphis japonica experienced two polyploidization events: the ancient whole genome triplication event shared with most eudicots (commonly known as the γ event) and a more recent whole genome duplication event, unique to E. japonica. By resequencing 101 samples from 11 populations, we speculate that the temperature has led to the differentiation of the evergreen and deciduous of E. japonica and the completely different population histories of these two groups. In total, 1012 candidate positively selected genes in the evergreen were detected, some of which are involved in flower and fruit development. We found that reddening and dehiscence of the E. japonica pericarp and long fruit-hanging time promoted the reproduction of E. japonica populations, and revealed the expression patterns of genes related to fruit reddening, dehiscence and abscission. The key genes involved in pentacyclic triterpene synthesis in E. japonica were identified, and different expression patterns of these genes may contribute to pentacyclic triterpene diversification. Our work sheds light on the evolution of E. japonica and malvids, particularly on the diversification of E. japonica and the genetic basis for their fruit dehiscence and abscission.

Genome-Wide Identification and Co-Expression Analysis of ARF and IAA Family Genes in Euscaphis konishii: Potential Regulators of Triterpenoids and Anthocyanin Biosynthesis.

Euscaphis konishii is an evergreen plant that is widely planted as an industrial crop in Southern China. It produces red fruits with abundant secondary metabolites, giving E. konishii high medicinal and ornamental value. Auxin signaling mediated by members of the AUXIN RESPONSE FACTOR (ARF) and auxin/indole-3-acetic acid (Aux/IAA) protein families plays important roles during plant growth and development. Aux/IAA and ARF genes have been described in many plants but have not yet been described in E. konishii. In this study, we identified 34 EkIAA and 29 EkARF proteins encoded by the E. konishii genome through database searching using HMMER. We also performed a bioinformatic characterization of EkIAA and EkARF genes, including their phylogenetic relationships, gene structures, chromosomal distribution, and cis-element analysis, as well as conserved motifs in the proteins. Our results suggest that EkIAA and EkARF genes have been relatively conserved over evolutionary history. Furthermore, we conducted expression and co-expression analyses of EkIAA and EkARF genes in leaves, branches, and fruits, which identified a subset of seven EkARF genes as potential regulators of triterpenoids and anthocyanin biosynthesis. RT-qPCR, yeast one-hybrid, and transient expression analyses showed that EkARF5.1 can directly interact with auxin response elements and regulate downstream gene expression. Our results may pave the way to elucidating the function of EkIAA and EkARF gene families in E. konishii, laying a foundation for further research on high-yielding industrial products and E. konishii breeding.

The re-sequencing of complete chloroplast genome of Cinnamomum camphora (Lauraceae) from Quanzhou, China.

Cinnamomum camphora is a precious species for its significance of timber. It is also the main component of the subtropical evergreen broad-leaved forest. We report a chloroplast genome of C. camphora from Quanzhou, China. The complete chloroplast genome is 152,730 bp in length consisting of a pair of inverted repeat (IR) regions of 19,968 bp, a small single-copy (SSC) region of 18,874 bp, and a large single-copy (LSC) region of 93,706 bp, respectively. The chloroplast genome encoded 116 genes, including 79 protein-coding genes, 36 tRNA genes, and eight rRNA genes. This result will provide useful genomic basis for the phylogenetic research of Lauraceae.

Total phenolic extract of Euscaphis konishii hayata Pericarp attenuates carbon tetrachloride (CCl4)-induced liver fibrosis in mice.

Liver fibrosis is a crucial pathological process involved in the hepatogenic morbidity and mortality. The pericarp of Euscaphis konishii Hayata is usually used in the cooking soup to improve liver function in Southern China, and high level of phenolic compounds has been found in the E. konishii pericarp. The total phenolic compounds extracted from E. konishii pericarp (TPEP) was obtained by polyamide column chromatograph, and 9 phenolic compounds of TPEP were identified through LC/MS and NMR. TPEP exhibited strong free radicals scavenging activity in vitro, and the chronic CCl4-induced liver fibrosis mice were established to elucidate the hepatoprotective mechanism of TPEP in vivo. TPEP treatment (50, 100 and 200 mg/kg) ameliorated the oxidative stress, immune dysfunction, inflammatory response and hepatic fibrosis induced by CCl4 introduction, alleviated the histopathological alteration and hepatocyte apoptosis in the liver tissue. Pretreatment with TPEP suppressed the activation of NADPH oxidase (NOX) signaling to attenuate oxidative stress in the liver tissue. TPEP administration inhibited the translocation of NF-κB into the nucleus to prevent the expression of downstream proinflammatory cytokines. TPEP treatment downregulated the activation of TGF-ß/Smad pathway, and facilitated the degradation of extracellular matrix through enhancing matrix metalloproteinases (MMPs) activity and decreasing the expression of matrix metalloproteinase inhibitors (TIMPs). In conclusion, TPEP inhibited CCl4-induced hepatic fibrosis through its antioxidant, anti-inflammatory and anti-fibrotic activities.

The complete chloroplast genome sequence of Tapiscia sinensis (Staphyleaceae).

Tapiscia sinensis, belong to Tapisciaceae, is endangered tree endemic to China. Here, we provide the complete plastid genomic data of T. sinensis with the aim of providing data for future conservation efforts research and revealing its phylogenetic position. The complete chloroplast sequence is 161,093 bp, including a large single-copy (LSC) region of 87,782 bp, a small single-copy (SSC) region of 18,517 bp, a pair of invert repeats (IR) regions of 27,387 bp. Plastid genome contains 131 genes, 85 protein-coding genes, 38 tRNA genes, and eight rRNA genes. Phylogenetic analysis base on 19 plastid genomes indicates that T. sinensis located Malvids branch, and is more closely related to the species of the order Sapindales than those of the order Malvales.

Protective Effect of the Total Triterpenes of Euscaphis konishii Hayata Pericarp on Bacillus Calmette-Guérin Plus Lipopolysaccharide-Induced Liver Injury.

BACKGROUND: Liver injury has been recognized as a primary cause of hepatic morbidity and mortality. Euscaphis konishii Hayata, also called Euscaphis fukienensis Hsu, is usually used as a detumescent and analgesic agent to improve liver function in South China, but its mechanism of action and chemical composition are unclear. OBJECTIVE: The main aim of the study was to investigate the constituent and potential hepatoprotective mechanism of the total triterpenes of E. konishii pericarp (TTEP). METHODS: The constituent of TTEP was analyzed by a series of silica gel column to get single compounds and then identified by NMR and MS. In vitro assays were conducted to test the free radical scavenging activity of TTEP. The BCG/LPS-induced immunological livery injury mice model was established to clarify the hepatoprotective effect of TTEP in vivo. RESULTS: 8 pentacyclic triterpene acids were separated and identified by NMR and MS. TTEP treatment (50, 100, and 200 mg/Kg) improved the immune function of the BCG/LPS-infected mice, dose-dependently alleviated the BCG/LPS-induced inflammation and oxidative stress, and ameliorated the hepatocyte apoptosis in the liver tissue. CONCLUSION: The pericarp of E. konishii may be further considered as a potent natural food for liver disease treatment.

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.

The complete chloroplast genome sequence of Brasenia schreberi (Cabombaceae).

Brasenia schreberi is a perennial aquatic herb. We determined the complete chloroplast genome sequence for B. schreberi using Illumina sequencing data. The complete chloroplast sequence is 158,993 bp, include large single-copy (LSC) region of 88,779 bp, small single-copy (SSC) region of 21,665 bp, a pair of invert repeats (IR) regions of 21,078 bp. Plastid genome contains 132 genes, 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis base on 12 chloroplast genomes indicates that B. schreberi is sister to Cabomba spp. in Cabombaceae.

Complete chloroplast genome sequence of Bougainvillea Spectabilis (Nyctaginaceae).

Bougainvillea spectabilis is an important ornamental plant in tropical and subtropical regions, whose complex and diverse germplasm has brought great difficulties to variety selection and identification. Here, to provide data support for the identification of the relationship between cultivars of B. spectabilis, we sequenced the chloroplast genome of B. spectabilis. The chloroplast genome is 154,520 bp in length, including a large single-copy (LSC) region of 88,101 bp and small single-copy (SSC) region of 17,729 bp, and a pair of invert repeats (IR) regions of 24,171 bp. The chloroplast genome contains 129 genes, including 83 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The phylogenetic analysis based on 14 chloroplast genome demonstrated a close relationship between B. spectabilis and other plant species in Caryophyllales. The chloroplast genome will help for further of taxonomy research and provide data support for the identification of genetic relationship among different cultivars of B. spectabilis.

Sequencing of Euscaphis konishii Endocarp Transcriptome Points to Molecular Mechanisms of Endocarp Coloration.

Flower and fruit colors are of vital importance to the ecology and economic market value of plants. The mechanisms of flower and fruit coloration have been well studied, especially among ornamental flower plants and cultivated fruits. As people pay more attention to exocarp coloration, the endocarp coloration in some species has often been ignored. Here, we report on the molecular mechanism of endocarp coloration in three development stages of Euscaphis konishii. The results show that endocarp reddening is closely related to anthocyanin accumulation, and a total of 86,120 unigenes were assembled, with a mean length of 893 bp (N50 length of 1642 bp). We identified a large number of differentially expressed genes associated with endocarp coloration, including anthocyanin biosynthesis, carotenoid biosynthesis, and chlorophyll breakdown. The genes participating in each step of the anthocyanin biosynthesis were found in the transcriptome dataset, but a few genes were found in the carotenoid biosynthesis and chlorophyll breakdown. In addition, the candidate R2R3-MYB transcription factors and candidate glutathione S-transferase transport genes, which likely regulate the anthocyanin biosynthesis, were identified. This study offers a platform for E. konishii functional genomic research and provides a reference for revealing the regulatory mechanisms of endocarp reddening.

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.

[Genetic relationships of Atractylodes plants].

The phylogenetic relationships of the genus Atractylodes DC. was analyzed using the nuclear ribosomal ITS and three chloroplast fragments, including atpB-rbcL, psbB-psbF and trnL-trnF intergenic spacer (IGS) sequences. Phylogenetic analyses revealed that A. lancea subsp. luotianensis and A. lancea, A. chinensis var. liaotungensis and A. coreana form monophyletic terminal clade, separately. The trees, within each the pairwise genetic distances, did not support Hu's classification. Authors suggested that A. lancea Subsp. luotianensis should be included in A. lancea rather than be treated as a separate subspecies. A. carlinoides was placed in the basal position of Atractylodes, which had a distant relationship with the others of the genus. The results lead us to suggest that A. chinensis var. liaotungensis be put into A. coreana, A. chinensis as a subspecies of A. lancea.