Swertiamarin attenuates carbon tetrachloride (CCl4)-induced liver injury and inflammation in rats by regulating the TLR4 signaling pathway

The aim of the present study is to illustrate the effects of swertiamarin (STM), a natural iridoid from herbal medicines, on hepatic inflammation induced by carbon tetrachloride (CCl4) in rats. Male Sprague Dawley rats were exposed to CCl4 with or without STM co-administration for 8 weeks. Our results revealed that STM administration (100 and 200 mg/kg b.w.) significantly attenuated inflammation in livers of CCl4-treated rats. STM remarkably reduced the production of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-1a (MIP-1α), and monocyte chemotactic protein-1 (MCP-1) in liver tissue of CCl4-treated rats. In addition, STM treatment downregulated connective tissue growth factor (CTGF) and ser307pIRS-1 expression, which was induced by CCl4 exposure. In the process of exploring the anti-inflammatory mechanisms of STM action, we demonstrated that STM significantly inhibited Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-κB) p65 expression in the liver. In conclusion, these results suggested that the inhibition of CCl4-induced inflammation by STM was, at least in part, due to its regulation of the TLR4 /NF-κB signaling pathway

Phylogeny of Helieae (Gentianaceae): Resolving taxonomic chaos in a Neotropical clade.

The monophyletic and Neotropical tribe Helieae of the worldwide family Gentianaceae (Gentianales, Asterids, Angiospermae) is well known for its problematic generic classifications. An initial phylogenetic analysis of Helieae shed light onto the relationships between genera, and indicated that traditional generic limits did not correspond to monophyletic groups. In order to obtain a more thorough understanding of generic relationships within the group, we enhanced sampling within the so-called Symbolanthus clade and performed phylogenetic analyses from DNA sequences from one plastid region (matK) and two nuclear regions (ITS and 5S-NTS), plus 112 morphological characters, which were analyzed separately and in combination, using parsimony and Bayesian approaches. A total of 83 individuals representing 20 genera and 51 species of Helieae were sampled; 13 species were included in this study solely based on their morphological characters. Ancestral character reconstructions were performed to identify potential synapomorphies of clades and patterns of homoplasy in the morphological dataset. Our results demonstrate that Prepusa is sister to the remainder of Helieae. Furthermore, the Macrocarpaea clade, the Irlbachia clade and the Symbolanthus clade were also recovered. Within the Symbolanthus clade, our results confirm that Calolisianthus and Chelonanthus are not monophyletic, and also contest the monophyly of Irlbachia as currently circumscribed. Specifically, two species of Calolisianthus group with the type species of Chelonanthus, while the other Calolisianthus species are more closely related to Tetrapollinia and Symbolanthus. Moreover, the green-white-flowered Chelonanthus species and Adenolisianthus are undoubtedly related to Helia and several analyses support Irlbachia pratensis as more closely related to the lineage including the type species of Chelonanthus described above The addition of new characters and taxa led to higher confidence in the relative position of some clades, as well as provided further support for a new generic circumscription of Calolisianthus, Chelonanthus, and Helia. Even though several morphological characters traditionally used in the taxonomy of the group were shown to be homoplasious, most clades can be diagnosed by a combination of morphological character states.

The Use of DNA Barcoding on Recently Diverged Species in the Genus Gentiana (Gentianaceae) in China.

DNA barcoding of plants poses particular challenges, especially in differentiating, recently diverged taxa. The genus Gentiana (Gentianaceae) is a species-rich plant group which rapidly radiated in the Himalaya-Hengduan Mountains in China. In this study, we tested the core plant barcode (rbcL + matK) and three promising complementary barcodes (trnH-psbA, ITS and ITS2) in 30 Gentiana species across 6 sections using three methods (the genetic distance-based method, Best Close Match and tree-based method). rbcL had the highest PCR efficiency and sequencing success (100%), while the lowest sequence recoverability was from ITS (68.35%). The presence of indels and inversions in trnH-psbA in Gentiana led to difficulties in sequence alignment. When using a single region for analysis, ITS exhibited the highest discriminatory power (60%-74.42%). Of the combinations, matK + ITS provided the highest discrimination success (71.43%-88.24%) and is recommended as the DNA barcode for the genus Gentiana. DNA barcoding proved effective in assigning most species to sections, though it performed poorly in some closely related species in sect. Cruciata because of hybridization events. Our analysis suggests that the status of G. pseudosquarrosa needs to be studied further. The utility of DNA barcoding was also verified in authenticating 'Qin-Jiao' Gentiana medicinal plants (G. macrophylla, G. crassicaulis, G. straminea, and G. dahurica), which can help ensure safe and correct usage of these well-known Chinese traditional medicinal herbs.

[A strategy for identifying six species of Sect.Cruciata(Gentiana) in Gansu using DNA barcode sequences].

Located in the transition zone between the Qinghai-Tibet Plateau and the Loess Plateau, Gansu province is one of the distribution centers of Sect. Cruciata, Gentiana (Gentianaceae) in China. Six species in the section, G. crassicaulis, G. straminea, G. siphonantha, G. officinalis, G. dahurica and G . macrophylla, are native to Gansu. In this paper, samples of 6 species and Halenia elliptica (outgroup) were collected. Nuclear DNA ITS, chloroplast DNA matK, rbcL, rpoC1, trnL (UAA) intron, psbA-trnH, atpB-rbcL, trnS (GCU)-trnG (UCC), rpl20-rps12 and trnL (UAA)-trnF (GAA) were sequenced from these samples. Based on the sequence analyses, high intragenomic polymorphisms were detected in ITS regions of G. crassicaulis, G. straminea, G. siphonantha, G. officinalis and G. dahurica, and they showed incomplete concerted evolution. A methodological study to identifying such close-related species as G. macrophylla, G. officinalis and G. dahurica was carried out based on the special genotypes. The results showed that 7 cp DNA sequence fragments could be used to identify G. crassicaulis, G. straminea and G. siphonantha. With nr ITS genotype II,III and IV of G. dahurica, the species can be distinguished from the close-related G. officinalis using 12 cloned sequences in a sample (with statistical significance).The cp DNA sequences of G. macrophylla were classified into two genotypes, and with genotype II, the species can be distinguished from the close-related G. officinalis and G. dahurica using 6 test samples each(with statistical significance). Furthermore, DNA barcode sequences were determined for all 6 species in Gansu. Also, the studies provide some basic data for analyses of genetic diversity and identification of Gentiana species.

[Study on identification of four kinds of Gentianaceae Mongolian medicine Digeda with spectroscopy techniques].

To study the identification of Gentianaceae Mongolian medicine Digeda with spectroscopy techniques, near infrared spectroscopy and differential scanning calorimetry techniques were applied to study on the identification of 4 kinds of Gentianaceae Mongolian medicine Digeda, and characteristic spectrums obtained were systematically analyzed. In NIR study, the four species of Digeda exist some differences in 4 250-4 400 cm(-1) and 5 650-5 800 cm(-1) of one-dimensional spectra, and show significant differences in 4 100- 4 400 cm(-1), 4 401-4 900 cm(-1) and 5 400-5 800 cm(-1) of the second derivative spectra. DSC curves of them present distinct topological pattern, characteristic peak and peak temperature. Using near infrared spectroscopy and differential scanning calorimetry analysis can realize efficient and accurate identification of four kinds of Mongolian medicine Digeda, and provide scientific basis for the efficient and accurate identification of other Gentianaceae Mongolian medicine Digeda.

[Preliminary comparative study of swertiamarin and swertisin on three kinds of Digeda-species Mongolian medicinal materials].

Lomatogonium rotatum (L.) Fries, Gentianopsis barbata (Froel) Ma, and Gentianella acuta (Michx.) Hulten, the three kinds of Digeda-species Mongolian medicinal materials belonging to the family Gentianaceae, bad been widely used for the treatment of liver diseases. To analyze comparatively the content of swertiamarin and swertisin among these three kinds of Digeda-species Mongolian medicinal materials. HPLC method was applied for qualitative and quantitative analysis of swertiamarin and swertisin. The Phenomenex C18 (4.6 mm x 250 mm, 5 µm) was used, chromatographic methanol and water as mobile phase, the flow rate was 1.5 mL x min(-1) with UV detected at 237 nm, column oven temperature was 25 degrees C. Results showed that the contents of swertiamarin and swertisin were closely related the different species and producing areas. The content range of swertiamarin in L. rotatum from different habitats was 1.73% - 2.72%, 0.43% - 0.96% for the swertisin content; the content of swertiamarin in G. barbata from Alxa Left Banner was 0.38%, and the content of swertiamarin and swertisin in G. barbata from the others habitats and G. Acuta from different habitats were all detected qualitatively. The contents of swertiamarin and swertisin among these medicinal plants showed a significant difference due to the different species and producing areas. As a consequence, these medicinal plants should not be put together for clinical applications.

Back to Gondwanaland: can ancient vicariance explain (some) Indian Ocean disjunct plant distributions?

Oceans, or other wide expanses of inhospitable environment, interrupt present day distributions of many plant groups. Using molecular dating techniques, generally incorporating fossil evidence, we can estimate when such distributions originated. Numerous dating analyses have recently precipitated a paradigm shift in the general explanations for the phenomenon, away from older geological causes, such as continental drift, in favour of more recent, long-distance dispersal (LDD). For example, the 'Gondwanan vicariance' scenario has been dismissed in various studies of Indian Ocean disjunct distributions. We used the gentian tribe Exaceae to reassess this scenario using molecular dating with minimum (fossil), maximum (geological), secondary (from wider analyses) and hypothesis-driven age constraints. Our results indicate that ancient vicariance cannot be ruled out as an explanation for the early origins of Exaceae across Africa, Madagascar and the Indian subcontinent unless a strong assumption is made about the maximum age of Gentianales. However, both the Gondwanan scenario and the available evidence suggest that there were also several, more recent, intercontinental dispersals during the diversification of the group.

Biosystematic studies on Enicostema axillare (Lam.) A. Raynal subsp. Axillare (Gentianaceae) in peninsular India.

The pantropical genus Enicostema (Gentianaceae) has three species and two sub species world over, namely, E. verticillatum (L.) Engl. (America), E. elizabethae Veldkamp (Madagascar) and E. axillare having 3 subsp. viz., subsp. axillare (Lam.) A. Raynal (India), subsp. latilobum (N.E. Br.) A. Raynal (East Africa) and subsp. littorale (Blume) A. Raynal (Indonesia). The present study aims to delimit the Indian taxa based on field and herbarium studies. Comparative morphology is studied using live as well as consulting wide range of specimens housed at various herbaria. The anatomy of leaf, stem, and root is studied using free hand sections and from epidermal peelings. The seed and pollen morphology are studied under SEM. Information on anatomy, palynology and seed micromorphology of E. axillare subsp. axillare is provided for the first time.

[Difference of chemical constituents contained in Tibetan herb Jiadiranguo from different habitats by HPLC fingerprint].

OBJECTIVE: To establish the HPLC fingerprint for Halenia elliptica herbs, a traditional Tibetan medicine, in order to study constituents contained in H. elliptica from different habitats and compare their differences. METHOD: HPLC analysis was made on a Welchrom-C18 (4.6 mm x 250 mm, 5 microm) with water and acetonitrile as mobile phase. The wavelength was detected as 265 nm, the flow rate was 1.0 mL x min(-1), and the column temperature was 40 degrees C. The software for chromatographic fingerprint was applied to analyze the similarity. And principal component analysis was conducted. RESULT: Twelve common chromatographic peaks were identified by fingerprint, showing a low similarity in constituent and variety. The significant difference in the proportion between xanthones and aglycones in each batch of herbs indicated no notable correlation between constituent characteristics and geographic locations of habitats. CONCLUSION: The method is so simple, exclusive, stable and highly repeatable that it can provide reference for identification and quality assessment of H. elliptica herbs.

Intraspecific inversions pose a challenge for the trnH-psbA plant DNA barcode.

BACKGROUND: The chloroplast trnH-psbA spacer region has been proposed as a prime candidate for use in DNA barcoding of plants because of its high substitution rate. However, frequent inversions associated with palindromic sequences within this region have been found in multiple lineages of Angiosperms and may complicate its use as a barcode, especially if they occur within species. METHODOLOGY/PRINCIPAL FINDINGS: Here, we evaluate the implications of intraspecific inversions in the trnH-psbA region for DNA barcoding efforts. We report polymorphic inversions within six species of Gentianaceae, all narrowly circumscribed morphologically: Gentiana algida, Gentiana fremontii, Gentianopsis crinita, Gentianopsis thermalis, Gentianopsis macrantha and Frasera speciosa. We analyze these sequences together with those from 15 other species of Gentianaceae and show that typical simple methods of sequence alignment can lead to misassignment of conspecifics and incorrect assessment of relationships. CONCLUSIONS/SIGNIFICANCE: Frequent inversions in the trnH-psbA region, if not recognized and aligned appropriately, may lead to large overestimates of the number of substitution events separating closely related lineages and to uniting more distantly related taxa that share the same form of the inversion. Thus, alignment of the trnH-psbA spacer region will need careful attention if it is used as a marker for DNA barcoding.

The polyphyletic genus Sebaea (Gentianaceae): a step forward in understanding the morphological and karyological evolution of the Exaceae.

Within the Gentianaceae-Exaceae, the most species-rich genus Sebaea has received very little attention in terms of phylogenetic or karyological investigations. As a result, the exact number of species remains vague and the relationships with the other members of the Exaceae poorly understood. In this paper, we provide the first comprehensive phylogeny of the Exaceae including most Sebaea species known so far based on four cpDNA sequence regions. In addition, morphological and karyological characters were mapped on the inferred phylogenetic trees to detect possible non-molecular synapomorphies. Our results reveal the paraphyly of Sebaea and highlight new generic relationships within the Exaceae. Sebaea pusilla (lineage S1--Lagenias) forms a highly supported and early diverging clade with Sebaeas.str. (clade S2 -Sebaea). A third clade of the former Sebaea s.l. (clade S3--Exochaenium) contains exclusively tropical African species, and is sister with a large clade containing all the remaining genera of Exaceae. Within the latter, the proposed sister relationships between the recently described Klackenbergia and Ornichia are highly supported. Optimization of several morphological characters onto the inferred phylogenetic trees reveals several synapomorphies for most highly supported clades. In particular, lineage S1 (Lagenias) is supported by medifixed anthers that are inserted at the base of the corolla tube and cubical seeds with polygonal testa cells; clade S2 (Sebaea) is supported by both the presence of secondary stigmas along the style and ridged seeds with rectangular testa cells arranged in row; clade S3 (Exochaenium) is supported by its particular gynoecium (stylar polymorphism and clavate, papillose stigma). Finally, karyological reconstructions suggest a basal number of x=7 for the Exaceae and several episodes of dysploidy leading to x=8 and 9.

Ultra-performance liquid chromatography/time-of-flight mass spectrometry as a chemotaxonomic tool for the analysis of Gentianaceae species.

INTRODUCTION: The segregation between the genera Gentiana and Gentianella among the Gentianaceae family is poorly defined. In order to clarify the classification of these genera, some researchers have tried to incorporate data about the chemical constitution, but this has not yet been achieved in a comprehensive way. OBJECTIVE: To develop a fast and reproducible analytical method for the observation of characteristic fingerprints of secondary metabolites of each genus. METHODOLOGY: Seven species were investigated, three Gentianella and four Gentiana selected for their close taxonomic links within each genus. Ten xanthones previously isolated from one of these species were used as chemotaxonomic markers. A UPLC/ESI-TOF-MS method was developed to analyse the methanolic extracts. RESULTS: The UPLC/TOF-MS provided clear metabolic fingerprints and elemental composition of the compounds. The profiles of the three Gentianella species were strikingly similar. On the contrary, metabolic profiles of Gentiana species were very different from the Gentianella chromatograms and also from each other. Several compounds were unique to each genus and therefore could be used as biomarkers. CONCLUSION: UPLC/TOF-MS can be applied as a chemotaxonomic tool for the rapid screening of Gentianaceae species and for the distinction between closely related taxa from the Gentianaceae family.

[Study on HPLC fingerprint of Gentiana macrophylla].

OBJECTIVE: To establish HPLC fingerprint of Gentiana macrophylla, which was expected to be standards of quality control and identification of the Chinese crude drug. METHODS: The HPLC method was used, chromatography conditions were Shimadzu C18 (150 mm x 4.6 mm, 5 microm) column with gradient mobile phase of methanol-0.4% Phosphoric acid, UV detection wavelength was at 230 nm and the column temperature was at 25 degrees C with the flow rate of 1.0 ml/min. RESULTS: 5 peaks were identified as the characteristic fingerprints of Gentiana macrophylla and the fingerprints of the 14 samples had high similarities. The similarities of disqualified and fake medicinal materials were low. CONCLUSION: This HPLC fingerprint of Gentiana macrophylla can be used as a standard of quality control and identification of the Chinese crude drug.

Molecular systematics and biogeography of Crawfurdia, Metagentiana and Tripterospermum (Gentianaceae) based on nuclear ribosomal and plastid DNA sequences.

BACKGROUND AND AIMS: The systematic position of the genus Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae. The aim of this paper is to examine the systematic position of Crawfurdia, Metagentiana and Tripterospermum and to clarify their phylogenetic affinities more clearly using ITS and trnL intron sequences. METHODS: Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the plastid DNA trnL (UAA) intron were analysed phylogenetically. Ten of fourteen Metagentiana species were sampled, together with 40 species of other genera in the subtribe Gentianinae. KEY RESULTS: The data support several previously published conclusions relating to the separation of Metagentiana from Gentiana and its closer relationships to Crawfurdia and Tripterospermum based on studies of gross morphology, floral anatomy, chromosomes, palynology, embryology and previous molecular data. The molecular clock hypothesis for the tested sequences in subtribe Gentianinae was not supported by the data (P < 0.05), so the clock-independent non-parametric rate smoothing method was used to estimate divergence time. This indicates that the separation of Crawfurdia, Metagentiana and Tripterospermum from Gentiana occurred about 11.4-21.4 Mya (million years ago), and the current species of these three genera diverged at times ranging from 0.4 to 6.2 Mya. CONCLUSIONS: The molecular analyses revealed that Crawfurdia, Metagentiana and Tripterospermum do not merit status as three separate genera, because sampled species of Crawfurdia and Tripterospermum are embedded within Metagentiana. The speciation and rapid radiation of these three genera is likely to have occurred in western China as a result of upthrust of the Himalayas during the late Miocene and the Pleistocene.

Morphological and ultrastructural diversity of orbicules in Gentianaceae.

Minute granules of sporopollenin, called orbicules, can be observed on the innermost tangential and/or radial walls of secretory tapetum cells. Orbicules were investigated in 53 species of 34 Gentianaceae genera using light microscopy, scanning electron microscopy and transmission electron microscopy. This selection covered all different tribes and subtribes recognized in Gentianaceae (87 genera, +/-1650 species). Orbicules were found in 38 species (23 genera) distributed among the six tribes recognized in Gentianaceae. The orbicule typology is based on those described previously in Rubiaceae. Of the six orbicule types described previously, Type II orbicules are lacking. Type III orbicules are most common (17 species). Hockinia Gardner is the only representative with Type I orbicules. The number of representatives with orbicules belonging to the other orbicule types are equally distributed among the species studied: seven species possess Type IV orbicules, six species Type V and six species Type VI. The systematic usefulness of this typology is discussed in comparison with the latest systematic insights within the family, and palynological trends in Gentianaceae. Orbicule data have proven to be useful for evaluating tribal delimitation within Rubiaceae and Loganiaceae s.l.; however, they seem not to be useful for tribal delimitation in Gentianaceae. In the tribes Potalieae and Gentianeae orbicule data may be useful at subtribal level.