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.

[Chemical constituents from the leaves of Cinnamomum camphora var. linaloolifera and their anti-inflammatory activities].

Thirteen compounds were isolated and purified from the leaves of Cinnamomum camphora by the macroporous resin,silica gel,and Sephadex LH-20 column chromatographies. Those compounds were further identified by IR,UV,MS,and NMR techniques:( 2 S)-1-( 3″,4″-methylenedioxy phenyl)-3-( 2',6'-dimethoxy-4'-hydroxyphenyl)-propan-2-ol( 1),( 2 R,3 R)-5,7-dimethoxy-3',4'-methylenedioxy flavanol( 2),9-hydroxysesamin( 3),sesamin( 4),piperitol( 5),kobusin( 6),(-)-aptosimon( 7),acuminatolide( 8),1ß,11-dihydroxy-5-eudesmene( 9),lasiodiplodin( 10),vanillin( 11),p-hydroxybenzaldehyde( 12),and p-hydroxybenzoic acid ethyl ester( 13). Compound 1 was a novel compound,and compounds 2,6,7,9 and 10 were isolated from Cinnamomum plants for the first time. Compounds 4,7 and 10 were found to possess good inhibitory effect on IL-6 production in LPS-induced BV2 cells at a concentration of 20 µmol·L-1 in the in vitro bioassay,with inhibition rates of 51. 26% ± 4. 13%,67. 82% ± 3. 77% and85. 81%±1. 19%,respectively.

[A new lignan from Euscaphis konishii].

The chemical constituents from the extract of the twigs of Euscaphis konishii with anti-hepatoma activity were investigated, twelve compounds by repeated chromatography with silica gel, Sephadex LH-20 and preparative-HPLC. The structures of the chemical components were elucidated by spectroscopy methods, as konilignan(1),(7R, 8S)-dihydrodehydrodico-niferylalcohol-9-O-ß-D-glucopyranoside(2),illiciumlignan B(3),threo-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-panediol(4),erythro-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-panediol(5), matairesinol(6), wikstromol(7), isolariciresinol(8),(+)-lyoniresinol(9), 4-ketopinoresinol(10), syringaresin(11), and vladinol D(12). Among them, compound 1 is a new lignan. Compounds 10 and 12 had moderate inhibitory activity on HepG2 cells, with IC_(50) values of 107.12 µmol·L~(-1) and 183.56 µmol·L~(-1), respectively.

Comprehensive transcriptome analysis of reference genes for fruit development of Euscaphis konishii.

BACKGROUND: Quantitativereal-time reverse transcriptase polymerase chain reaction is the common method to quantify relative gene expression. Normalizating using reliable genes is critical in correctly interpreting expression data from qRT-PCR. Euscaphis konishii is a medicinal plant with a long history in China, which has various chemical compounds in fruit. However, there is no report describing the selection of reference genes in fruit development of Euscaphis konishii. METHODS: We selected eight candidate reference genes based on RNA-seq database analysis, and ranked expression stability using statistical algorithms GeNorm, NormFinder, BestKeeper and ReFinder. Finally, The nine genes related to the anthocyanin synthesis pathway of Euscaphis konishii were used to verify the suitability of reference gene. RESULTS: The results showed that the stability of EkUBC23, EkCYP38 and EkGAPDH2 was better, and the low expression reference genes (EkUBC23 and EkCYP38) were favourable for quantifying low expression target genes, while the high expression reference gene (EkGAPDH2) was beneficial for quantifying high expression genes. In this study, we present the suitable reference genes for fruit development of Euscaphis konishii based on transcriptome data, our study will contribute to further studies in molecular biology and gene function on Euscaphis konishii and other closely related species.

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.

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 Euscaphis japonica (Staphyleaceae).

Euscaphis japonica is not only an ideal ornamental plant, but also a traditional medicinal plant, which is an extremely valuable species to study. We determined the complete chloroplast genome sequence for E. japonica using Illumina sequencing technology. The complete chloroplast sequence is 160,467 bp in length, including a large single-copy (LSC) region of 88,716 bp, a small single-copy (SSC) region of 18,614 bp, and a pair of invert repeats (IR) regions of 26,568 bp. Plastid genome contains 142 genes, 46 protein-coding genes, 39 tRNA genes, and eight rRNA genes. Phylogenetic analysis base on 14 chloroplast genomes indicates that E. japonica forms an isolated clade and sisters to Glycosmis-Gossypium clade with strong support.

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.