Identification of C-glycosyl flavones and quality assessment in Dendrobium nobile.

RATIONALE: Flavones are significant indicators of quality in traditional Chinese medicines (TCMs) and thus play a significant role in the quality control of TCMs in the pharmaceutical industry. Most flavones in Dendrobium nobile Lindl, a TCM with a long cultivation history and rich sources, have not been identified. This study was aimed at identifying the flavones in D. nobile from various habitats. METHODS: High-performance liquid chromatography (HPLC) coupled with diode-array detection and HPLC multiple-stage tandem mass spectrometry was used to identify the chemical constituents of D. nobile from various habitats, and a method was established to determine the content of vicenin II, violanthin and isoviolanthin. Hierarchical cluster analysis, principal component analysis and orthogonal partial least-squares discriminant analysis were used to analyze the variations among 26 batches from different habitats. RESULTS: A total of 33 flavones were tentatively identified. Twenty-five flavones, previously undescribed in D. nobile, were acylated by p-coumaroyl, feruloyl, sinapoyl or 3-hydroxy-3-methylglutaryl. The D. nobile habitats were distinguished by significant differences in their flavone content. The C-glycosyl flavones were demonstrated to be characteristic compounds for evaluating D. nobile from various habitats. In particular, flavones acylated with 3-hydroxy-3-methylglutaryl were specific compounds that were only detected in samples from Yunnan. CONCLUSIONS: The results of this study could be used to improve the quality control of D. nobile and could provide references for the identification of acylated C-glycosyl flavones in other natural products.

Functional analysis of a novel C-glycosyltransferase in the orchid Dendrobium catenatum.

Flavonoids, which are a diverse class of phytonutrients, are used by organisms to respond to nearly all abiotic stresses and are beneficial for human health. Glycosyltransferase, used during the last step of flavonoid biosynthesis, is important in flavonoid enrichment. However, little is known about glycosyltransferase in the orchid Dendrobium catenatum (D. officinale). In this study, we isolated a novel C-glycosyltransferase (designated DcaCGT) from the orchid D. catenatum by identifying and analyzing 82 putative genes in the GT1 family. DcaCGT could specifically catalyze not only di-C-glycosylation but also O-glycosylation. Apart from the normal function of catalyzing 2-hydroxynaringenin and phloretin to the respective di-C-glycosides, DcaCGT also catalyzes apigenin to cosmosiin. Targeted metabolic profiling of the substrates (2-hydroxynaringenin, phloretin, and apigenin) and products (vitexin, isovitexin, vicenin-2, nothofagin, 3',5'-di-C-glucosylphloretin, and cosmosiin) in different tissues showed that vicenin-2 was the most abundant product of this novel enzyme. Cosmosiin was detected in flowers and flower buds. We also established that DcaCGT functions expanded throughout the evolution of D. catenatum. Residual OGT activity may help D. catenatum resist drought stress. Our study illustrates the function, origin, and differentiation of DcaCGT and provides insights into glycosylation and molecular propagation processes, which can be used to improve the production of flavonoids by the cultivated medicinal plant D. catenatum.

Network Analysis of Transcriptome and LC-MS Reveals a Possible Biosynthesis Pathway of Anthocyanins in Dendrobium officinale.

Anthocyanins, a group of flavonoids, are widely present in plants and determine the colors of the peels of stems, fruits, and flowers. In this study, we used UHPLC-ESI-MS to identify anthocyanins in the herbal plant Dendrobium officinale, which has been used for centuries in China. The results indicated that the total anthocyanin content in samples from Guangxi was the highest. Seven anthocyanins were identified, and the fragmentation pathways were proposed from D. officinale. Most of the identified anthocyanins were composed of cyanidin and sinapoyl groups. We also carried out that the sinapoyl group had active sites on breast cancer receptors by using Schrödinger. The relative levels of the 7 anthocyanins in the samples from the three locations were determined. Transcriptomic analysis was used to analyze the sinapoyl anthocyanin synthesis-related genes in plants, such as genes encoding UGTs and serine carboxypeptidase. We speculated that sinapoyl anthocyanin biosynthesis was associated with the activities of certain enzymes, including chalcone flavonone isomerase-like, hydroxycinnamoyltransferase 1, UGT-83A1, UGT-88B1 isoform X1, serine carboxypeptidase-like 18 isoform X3, and serine carboxypeptidase-like 18.

[Cluster analysis of Dendrobium officinale from different populations through agronomy characterization].

Through resources investigation and sample collection,a total number of 392 Dendrobium officinale from 38 different populations,9 provinces were processed for measuring and observing.Fourteen agronomy characterizations like stem height,stem diameter,number of node in stem were selected for further classification.The cluster analysis was performed using Ward and Euclidean method.The results showed that the threshold of genetic distance was 2.77.Thirtyeight populations were divided into 6 groups.The factor analysis showed that,the leaf shape,stem shape,pitch length and leaf color were very important factors for classification.This study establish the foundation for analyzing the genetic relationship of D.officinale from different populations.

Structure Identification of ViceninII Extracted from Dendrobium officinale and the Reversal of TGF-ß1-Induced Epithelial⁻Mesenchymal Transition in Lung Adenocarcinoma Cells through TGF-ß/Smad and PI3K/Akt/mTOR Signaling Pathways.

ViceninII is a naturally flavonoid glycoside extracted from Dendrobium officinale, a precious Chinese traditional herb, has been proven to be valuable for cancer treatment. Transforming growth factor-ß1 (TGF-ß1), promotes the induction of epithelial⁻mesenchymal transition (EMT), a process involved in the metastasis of cells that leads to enhanced migration and invasion. However, there is no previously evidence that ViceninII has an inhibitory effect on cancer metastasis, specifically on the TGF-ß1-induced EMT process in lung adenocarcinoma cells. In this experiment, we used UV, ESIMS, and NMR to identify the structure of ViceninII.A549 and H1299 cells were treated with TGF-ß1 in the absence and presence of ViceninII, and subsequent migration and invasion were measured by wound-healing and transwell assays. The protein localization and expressions were detected by immunofluorescence and Western blotting. The results indicated that TGF-ß1 induced spindle-shaped changes, increased migration and invasion, and upregulated or downregulated the relative expression of EMT biomarkers. Meanwhile, these alterations were significantly inhibited when co-treated with ViceninII and inhibitors LY294002 and SB431542. In conclusion, ViceninII inhibited TGF-ß1-induced EMT via the deactivation of TGF-ß/Smad and PI3K/Akt/mTOR signaling pathways.This is the first time that the anti-metastatic effects of ViceninII have been demonstrated, and their molecular mechanisms provided.

Structural Characterization of Polysaccharides from Dendrobium officinale and Their Effects on Apoptosis of HeLa Cell Line.

Dendrobium officinale is a widely used medicinal plant in China with numerous bio-activities. However, the main structure and anti-tumor activity of the polysaccharides from this plant have not been investigated. In this study, we elucidated the main structure of polysaccharides purified with DEAE and Sephadex G-25 from Dendrobium officinale grown under different planting conditions. In addition, the anti-tumor activity was tested via MTT assays. The results showed that the polysaccharides of Dendrobium officinale grown under different conditions were almost the same, with slight differences in the branched chain; both polysaccharide fractions consisted of (1→4)-linked mannose and (1→4)-linked glucose, with an O-acetyl group in the mannose. After degradation, the polysaccharide fractions from wild plants showed significant anti-proliferation activity in HeLa cells. The fractions F1 and F3 induced apoptosis by up-regulating the expression of ERK, JNK, and p38. We concluded that polysaccharides from Dendrobium officinale planted in the wild exhibit significant anti-tumor effects only after being degraded to smaller molecular weight species. The planting mode is a significant factor in the pharmacological activity of Dendrobium officinale. We advise that the planting conditions for Dendrobium officinale should be changed.

UHPLC-ESI-MS Analysis of Purified Flavonoids Fraction from Stem of Dendrobium denneaum Paxt. and Its Preliminary Study in Inducing Apoptosis of HepG2 Cells.

Dendrobium denneaum paxt., which has been widely used for health prevention in traditional Chinese medicine (TCM), is one of the most popular tonic herbs in China. In order to analyze its flavonoids, characterization and antitumor activity of crude extract and flavonoids rich fractions from D. denneaum paxt. were investigated. Flavonoids extracted from D. denneaum paxt. were clearly enriched in fraction II after determining the total flavonoids content; there were 15 characteristic peaks which have been detected; ultra-high performance liquid chromatography-electrospray ionization/mass spectrometry (UHPLC-ESI-MS/MS) was applied for structural elucidation of compounds. 13 characteristic peaks including flavonoid-O-glycosides and flavonoid-C-glycosides were determined or preliminarily characterized through comparing retention times and UV and MS spectra with standard compounds or documented literature. The antitumor activity of fraction II on human liver cancer cells HepG2 was investigated. MTT assay method was used to test the antiproliferation activity and to confirm the appropriate treatment concentration as well as inducing time. The morphological changes of the apoptosis cells after being induced by fraction II were observed by a Hoechst reagent and the apoptosis rate was tested by flow cytometry. The results showed that fraction II can inhibit HepG2 cells from proliferation in a dose-dependent and time-dependent manner. The apoptosis experiments indicated that fraction II can significantly induce apoptosis in HepG2 cells in a concentration over 50 µg/mL for 48 h and the most effective level was 150 µg/mL for 48 h.

Isoviolanthin Extracted from Dendrobium officinale Reverses TGF-ß1-Mediated Epithelial⁻Mesenchymal Transition in Hepatocellular Carcinoma Cells via Deactivating the TGF-ß/Smad and PI3K/Akt/mTOR Signaling Pathways.

Dendrobium officinale is a precious medicinal herb and health food, and its pharmacological actions have been studied and proved. However, the mechanisms by which its active flavonoid glycosides affect epithelial⁻mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) cells, such as HepG2 and Bel-7402 cells, have not been previously investigated. Therefore, we investigated whether isoviolanthin extracted from the leaves of Dendrobium officinale inhibits transforming growth factor (TGF)-ß1-induced EMT in HCC cells. In this study, the physicochemical properties and structure of isoviolanthin were identified by HPLC, UV, ESIMS, and NMR and were compared with literature data. HCC cells were pretreated with 10 ng/mL TGF-ß1 to induce EMT and then treated with isoviolanthin. Herein, we found that isoviolanthin exhibited no cytotoxic effects on normal liver LO2 cells but notably reduced the migratory and invasive capacities of TGF-ß1-treated HCC cells. Additionally, isoviolanthin treatment decreased matrix metalloproteinase (MMP)-2 and -9 levels, and remarkably altered the expression of EMT markers via regulating the TGF-ß/Smad and PI3K/Akt/mTOR signaling pathways; Western blot analysis confirmed that the effects of the inhibitors SB431542 and LY294002 were consistent with those of isoviolanthin. These findings demonstrate the potential of isoviolanthin as a therapeutic agent for the treatment of advanced-stage metastatic HCC.

Simultaneous identification and determination of flavonoids in Dendrobium officinale.

BACKGROUND: The quality of material medicine resources has had a considerable impact on the development of the health industry, which has created a bottleneck for traditional Chinese medicine (TCM). Dendrobium officinale, which has been widely used for health prevention in TCM, has become a high-nutritive health food that is strongly recommended by many white-collar workers and people who pay more attention to their health. The aim of this study was to develop a method to authenticate and evaluate D. officinale from different origins via simultaneous qualitative and quantitative analyses of flavonoid glycosides. Ultra-high-performance liquid chromatography-electrospray ionization/mass spectrometry was used for the structural elucidation of the compounds. RESULTS: 9 characteristic peaks, including those representing 7 flavonoid C-glycosides and 2 flavonoid O-glycosides, were identified. Additionally, the contents of 5 representative flavonoid glucosides in 25 batches of D. officinale from different sources were determined. To further investigate the different sources of the 25 batch samples, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were carried out. A study on the methodology revealed that all results were reliable. CONCLUSIONS: This method is an efficient tool for the rapid identification of the different geographical origins of D. officinale and provides references for the quality evaluation of other natural products.

Transcriptome Analysis Reveals genes involved in flavonoid biosynthesis and accumulation in Dendrobium catenatum From Different Locations.

In this study, we applied transcriptome and UHPLC-MS technologies to investigate the flavonoids and their biosynthesis- and accumulation-related genes in Dendrobium catenatum from three different locations. Eight flavonoid glycosides were identified using standard references or previously isolated substances with MS data analysis. The total flavonoid contents were determined by reagents, and all the data were analyzed. In total, 23139 unigenes were obtained using the Dendrobium catenatum genome data. Of these, 10398 were annotated in the Gene Ontology (GO) database, 4203 were annotated in the KEGG database, and 10917 were annotated in the EuKaryotic Orthologous Groups (KOG) database. Thirty-one of the unigenes annotated by the KEGG database were involved in flavonoid pathways. The genes involved in bio-modification, accumulation, transportation and the regulation of the flavonoid bio-synthesis process were investigated. In conclusion, the flavonoids in Dendrobium catenatum from three different locations were different in quantitative and qualitative which may contribute to the establishment of quality control method for this herbal plant. These differences were determined by flavonoids biosynthesis process and they were concluded by sorting out the expression level of certain biosynthesis related genes.

Rosmarinic acid protects on rat bone marrow mesenchymal stem cells from hydrogen peroxide-induced apoptosis.

To investigate the anti-oxidant activities and mechanism of rosmarinic acid (RA) on rat bone marrow mesenchymal stem cells (rBMSCs) from ischemia-induced apoptosis in vitro, which was established using H2O2-damage and analyzed for cell viability, cell apoptosis, ROS, morphological changes, and levels of apoptosis proteins. Pretreatment with RA significantly suppressed the generation of ROS, protected the morphological changes of cells, decrease the ratio of cell apoptosis, down-regulated the level of caspase-3, caspase-9, Bax/Bcl-2, and up-regulated the level of p-PI3K. These findings suggest that RA may protect rBMSCs from H2O2-induced apoptosis by partly regulating PI3K/Akt signaling pathway and can be developed as a potential anti-apoptotic agent for therapy in cardiovascular diseases.