Phylogenetic analysis of chloroplast genome of Tussilago farfara L / 药学学报

italic>Tussilago farfara L. is a perennial herb of Tussilago genus in the Compositae family. Its dried buds and leaves have good biological activities and have a long history of medicinal use in China and Europe. In this paper, we investigated the whole chloroplast genome characteristics, sequence duplication, structural variation and phylogeny of the Tussilago farfara L. After sequencing the Tussilago farfara L. chloroplast genome using Illumination technology, the complete Tussilago farfara L. chloroplast genome was further obtained by assembly and annotation, followed by a series of inverted repeat-large single copy/small single copy region contraction and expansion analysis, genome sequence variation, etc. The sequences of 13 homologous plants downloaded from NCBI were used to construct a neighbor-joining phylogenetic tree. The results showed that the total GC content of the chloroplast genome was 37.4% and the length was 150 300 bp; 125 genes were annotated, including 82 protein-coding genes, 35 tRNAs and 8 rRNAs; 148 (simple sequence repeats, SSR) loci were detected, and the relative synonymous codon usage showed that 31 codons out of 64 codons had a usage of >1. In the phylogenetic analysis, the chloroplast genomes of the seven species of Asteraceae, including the Yulin Tussilago farfara L., were highly conserved, and the sequence variation of the (large single-copy, LSC) and (small single-copy, SSC) regions was higher than that of the (inverted repeat, IR) region. This is in general agreement with the reported phylogeny of Yulin Tussilago farfara L. In this study, we obtained a high quality chloroplast genome and analyzed its genome characteristics, codon preference, SSR characteristics, SC/IR boundary, sequence variation and phylogeny, which can provide a basis for species identification, genetic diversity analysis and resource development of this medicinal plant.

Polyhydroxylated eudesmane sesquiterpenoids and sesquiterpenoid glucoside from the flower buds of Tussilago farfara / 中国天然药物

Chemical fractionation of the n-BuOH partition, which was generated from the EtOH extract of the flower buds of Tussilago farfara, afforded a series of polar constituents including four new sesquiterpenoids (1-4), one new sesquiterpenoid glucoside (5) and one known analogue (6) of the eudesmane type, as well as five known quinic acid derivatives (7-11). Structures of the new compounds were unambiguously characterized by detailed spectroscopic analyses, with their absolute configurations being established by X-ray crystallography, electronic circular dichroism (ECD) calculation and induced ECD experiments. The inhibitory effect of all the isolates against LPS-induced NO production in murine RAW264.7 macrophages was evaluated, with isochlorogenic acid A (7) showing significant inhibitory activity.

Analysis of key genes and their expression characteristics related to terpenoid biosynthesis in Tussilago farfara based on transcriptome sequencing / 中草药

Objective: To screen candidate genes involved in the terpenoid biosynthetic pathway of Tussilago farfara. Methods: The transcriptome of buds and leaves of wild T. farfara were respectively sequenced using the Illumina HiSeq 2500 high-throughput sequencing platform. The clean reads were de novo assembled by Trinity software, and the assembled sequences then followed by a series of bioinformatics analysis such as gene function annotation and differential expression gene. According to sequence annotation and differentially expressed genes analysis, the key enzyme genes related to the terpenoid biosynthesis were identified. Results: After high through-put sequencing, a total of 39 912 371 clean reads were obtained (SRA accession: SRR9113366, SRR9113367). The clean reads were then assembled into 91 118 unigenes. A total of 55 830 unigenes were annotated by a similarity search against NR, Swiss-Port, GO, COG, KEGG five public databases. Base on KEGG annotation and differentially expressed genes, totally 129 catalytic enzyme genes referring to the terpenoid biosynthesis were identified, including 91 terpenoid backbone biosynthesis genes, 32 terpene synthases, and 6 cytochrome P450 (CYP450) genes. Among them, 25 genes were differentially expressed. The expression of four enzyme genes in MVA pathway in leaves were higher than that in buds, while the five enzyme genes in MEP pathway were lower in leaves than that in buds. In addition, 10 genes were highly expressed in leaves, and nine genes were highly expressed in buds. According to the high expression of differentially expressed HMGR, TPS, AS, CYP450 genes in buds, it was speculated that these genes may be related to the high content of terpenoids in flower buds. Conclusion: This work obtained candidate key enzyme genes that may be involved in the biosynthesis of terpenoid by transcriptome sequencing. The results laid a foundation for further elucidating the molecular mechanism of terpenoid biosynthetic pathway in T. farfara.

Analysis on liver and renal toxicity of leaves of Tussilago farfara using zebrafish / 中草药

Objective: To compare the toxicity of the flower buds and leaves from Tussilago farfara, and provide scientific basis for the utilization of the leaves of T. farfara. Methods: The 3 dpf (day post fertilization) healthy AB and transgene zebrafish were selected. The flowers, the leaves, the flowers coupled with Aster tataricus, and the leaves coupled with A. tataricus were prepared at the concentration of 0.5, 1.0, and 1.5 mg/mL. The fluorescent area of the liver and ALT and AST levels were measured after 72 h of drug treatment. For the renal toxicity assay, the morphology of zebrafish and the nutrient solution protein were also determined. Results: Compared with the control group, there were no significant differences in liver biochemical indexes in the four drug treatment groups. However, the fluorescence area of liver decreased in the flowers and flowers coupled with A. tataricus group at the concentration of 1.5 mg/mL. No significant difference was observed in the four groups of the nephrotoxicity assay. Conclusion: The flower and the flower coupled with A. tataricus showed minor hepatotoxicity at higher doses, and the leaves showed no stronger toxicity than the flower buds in comparison with the flower buds. It can provide refercences for the resource utilization of the leaves of T. farfara.

Development and polymorphism analysis of SSR molecular markers in Tussilago farfara transcriptome / 中草药

Objective: The SSR loci information in the transcriptome of Tussilago farfara was analyzed and specific primers were designed, so as to provide powerful tools for molecular marker-assisted breeding in this plant. Methods: SSR loci in 18 938 unigenes with length of 1 kb or more obtained by transcriptome sequencing were searched by using MISA. SSR primers were designed by Primer3 and 55 pairs were randomly selected for the polymorphic analysis on 18 samples collected from different habitats. Results: A total of 4 688 SSRs were detected in the transcriptome of T. farfara, distributed in 3 844 unigenes with the distribution frequency of 24.75%. SSR loci occurred every 7 979 bp. Trinucleotide repeats appeared to be the most abundant SSRs with a frequency of 37.12%, followed by mononucleotide (32.36%) and dinucleotide (28.20%). Among all 60 repeat motifs, A/T (31.42%), AG/CT (12.80%), and ATC/ATG (9.62%) were the predominant repeat types. For validating the availability of the SSR primers, 55 pairs of primers were randomly selected for polymorphism analysis. Among them, 42 pairs (76.36%) produced clear and reproductive bands and 14 pairs showed polymorphism. Eighteen plants were divided into three groups by UPGMA. Conclusion: The SSR markers in the transcriptome of T. farfara show high frequency, rich type, and high polymorphism, which will provide the abundant candidate markers for genetic diversity, genetic mapping construction and marker-assisted breeding study for this plant.

Study on Quality Standard for Tussilago farfara Formula Granules / 中国药房

OBJECTIVE： To provide reference for the establishment of quality standard of Tussilago farfara formula granules. METHODS： TLC method was used for qualitative identification of tussilagone in T. farfara formula granules. The content of tussilagone in T. farfara formula granule was determined by HPLC. The determination was performed on a Thermo ODS Hypersil C18 column with the mobile phase consisted of methanol-water （85 ∶ 15， V/V） at the flow rate of 1.0 mL/min. The detection wavelength was set at 220 nm， the column temperature was 25 ℃. Sample size was 20 μL.  RESULTS： TLC spots of tussilagone were clear and well-separated， without interference from negative control. The linear range of tussilagone was 1.39-27.75 μg/mL （r＝0.999 9）. The limits of quantification and detection were 0.153 87 and 0.051 42 μg/mL， respectively. RSDs of precision， stability and reproducibility tests were lower than 2％. The recoveries were 97.12％-103.96％ （RSD＝2.60％， n＝6）. CONCLUSIONS： The method is simple， accurate and reproducible， and suitable for quality control of T. farfara formula granules.

Chemical Comparison of the Stems and Leaves of Tussilago farfara L. Using NMR-based Metabolomics / 中国药学杂志

OBJECTIVE: To compare the chemical compositions of the stems (STL) and leaves (LTL) of Tussilago farfara L. using NMR-based metabolomic approach. METHODS: The STL and LTL were analyzed by NMR, then the differential components were determined by multivariate statistical METHODS:, including PCA, PLS-DA, OPLS-DA, and univariate analysis. RESULTS: Fifty compounds were tentatively identified in the NMR spectra of STL and LTL. Multivariate coupled with univariate analysis revealed that some metabolites, such as valine,leucine,isoleucine,proline,chlorogenic acid,3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, and tussilagone,were present at higher levels in the LTF, while the STF contained higher levels of α-glucose and β-glucose.For some metabolites, such as malic acid, sucrose and choline, no significant differences were observed between STF and LTF. CONCLUSION: This study reveals the chemical differences between STF and LTF in a holistic way, and lays the scientific foundation for the resource utilization of the stems and leaves of T. farfara L.

Comparison of flower buds and leaves of Tussilago farfara L. by UHPLC-Q Extractive Hybrid Quadrupole Orbitrap MS / 药学学报

The flower bud of Tussilago farfara L. has been commonly used in the treatment of cough, bronchitis and asthmatic disorders in the Traditional Chinese Medicine. In Europe, the leaves were also used as herbal drugs with similar pharmacological activities. In order to utilize the leaves, it is important to conduct the chemical comparison between the flower buds and the leaves. In this study, ultra high liquid chromatography (UHPLC) coupled with Q Exactive high resolution mass spectrometry (HR-MS) was used to compare the chemical composition of the flower buds and leaves of T. farfara L. forty three metabolites were identified by the combination of targeted and untargeted approach. The results suggest that the sesquiterpenes, such as tussilagone and 7β-(3'-ethyl-cis-crotonoyloxy)-1α-(2'-methylbutyryloxy)-3(14)-dehydro-Z-notonipetranone were higher in the flower buds. While the phenylpropanoids, such as cholorgenic acid and isochlorogenic acid were higher in the leaves. The flavonoids, such as hyperin and quercetin exhibited no difference between the flower buds and leaves, while the rutin and kaempferol were higher in the flower buds. The leaves and flower buds had similar chemical components, and the phenylpropanoids, which were closely related with the antitussive and expectorant activities, were found at higher concentrations in the leaves. The results presented here laid the basis for the rational utilization of the leaves of T. farfara L.

Effects of Flower Buds Extract of Tussilago farfara on Focal Cerebral Ischemia in Rats and Inflammatory Response in BV2 Microglia / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of the flower buds extract of Tussilago farfara Linné (Farfarae Flos; FF) on focal cerebral ischemia through regulation of inflammatory responses in activated microglia.</p><p><b>METHODS</b>Brain ischemia was induced in Sprague-Dawley rats by a transient middle cerebral artery occlusion (tMCAO) for 90 min and reperfusion for 24 h. Twenty rats were randomly divided into 4 groups (n=5 per group): normal, tMCAO-induced ischemic control, tMCAO plus FF extract 300 mg/kg-treated, and tMCAO plus MK-801 1 mg/kg-treated as reference drug. FF extract (300 mg/kg, p.o.) or MK-801 (1 mg/kg, i.p.) was administered after reperfusion. Brain infarction was measured by 2,3,5,-triphenyltetrazolium chloride staining. Neuronal damage was observed by haematoxylin eosin, Nissl staining and immunohistochemistry using anti-neuronal nuclei (NeuN), anti-glial fibrillary acidic protein (GFAP), and anti-CD11b/c (OX42) antibodies in ischemic brain. The expressions of inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF-α), and hypoxia-inducible factor-1a (HIF-1α) were determined by Western blot. BV2 microglial cells were treated with FF extract or its main bioactive compound, tussilagone with or without lipopolysaccharide (LPS). Nitric oxide (NO) production was measured in culture medium by Griess assay. The expressions of iNOS, COX-2 and pro-inflammatory cytokines mRNA were analyzed by reverse transcription-polymerase chain reaction. The expression of iNOS, and COX-2 proteins, the phosphorylation of ERK1/2, JNK, and p38 MAPK and the nuclear expression of NF-κB p65 in BV2 cells were determined by Western blot.</p><p><b>RESULTS</b>FF extract significantly decreased brain infarctions in ischemic rats (P<0.01). The neuronal death and the microglia/astrocytes activation in ischemic brains were inhibited by FF extract. FF extract also suppressed iNOS, TNF-α, and HIF-1α expression in ischemic brains. FF extract (0.2 and 0.5 mg/mL, P<0.01) and tussilagone 20 and 50 μmol/L, P<0.01) significantly decreased LPS-induced NO production in BV2 microglia through downregulation of iNOS mRNA and protein expression. FF extract and tussilagone significantly inhibited LPS-induced expression of TNF-α, IL-1β, and IL-6 mRNA, and also suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK and the nuclear expression of NF-κB in a dose-dependent manner.</p><p><b>CONCLUSIONS</b>FF extract has a neuroprotective effect in ischemic stroke by the decrease of brain infarction, and the inhibition of neuronal death and microglial activation-mediated inflammatory responses.</p>

Comparative transcriptomic analysis of leaves of Tussilago farfara in different development stages / 中草药

Objective: To compare the expression of genes in the leaves of Tussilago farfara that involved in biosynthesis of phenylpropanoids in different developmental stages, and infer the accumulation period of biosynthesis of phenylpropanoids and provide a scientific basis for the resource utilization of leaves of T. farfara. Methods: The Illumina HiSeq2500 highthroughput sequencing method was used to analyze the transcriptome of the leaves of T. farfara in different periods. After obtaining transcriptome data, bioinformatics analysis of gene function annotation was performed to compare the expression of genes related to phenylpropanoid biosynthesis in different periods. Results: A total of 46 793 unigenes were obtained by transcriptome sequencing and the average length was 952.144 8 bp. Among them, 4 774 unigenes were annotated in the public databases NR, Swiss-Prot, eggNOG, GO, and KEGG. According to the assignment of KEGG pathway, 144 unigenes were involved in terpenoid biosynthesis, phenylpropanoid biosynthesis and flavonoids, 65 unigenes were involved in terpenoid biosynthesis, 64 unigenes were involved in phenylpropanoid and 15 unigenes were involved in flavonoids biosynthesis. The enzyme genes involved in the phenylpropanoid biosynthesis were also compared in different development stages, and the results indicated that the expression of PAL, 4CL, HCT, and CCoAOMT, which were closely related to biosynthesis of phenylpropanoids, were highest in September, which means that the contents of these compounds might be highest in September. Conclusion: This study lays the foundation for the biosynthetic pathway and regulation analysis of phenylpropanoids, and provides a scientific basis for the development and the resource utilization of leaves of T. farfara.

Tissue culture of callus and establishment of regeneration system of Tussilago farfara petiole / 中国中药杂志

Young petiole of Tussilago farfara was used as the material to investigate the plant growth regulators which could influence in vitro culture and plant regeneration and to establish rapid propagation technique. The ideal sterilization method was that young petiole of T. farfara was sterilized with 75% ethanol for 30 s, and then transferred to saturated bleaching power supernatant for 15 min. The suitable medium for callus induction was MS+6-BA 3.0 mg•L⁻¹+2,4-D 2.0 mg•L⁻¹ with 96.2% induction rate. The seedlings had better differentiation with 91% differentiation rate and 8.26 buds on the medium containing MS+ZT 2.0 mg•L⁻¹+NAA 0.3 mg•L⁻¹. The preferred enrichment medium of adventitious bud was MS+KT 1.0 mg•L⁻¹+IBA 0.3 mg•L⁻¹ with 11.81 enrichment times and 4.9 cm seedling height. The rooting medium included 1/2MS+IBA 0.2 mg•L⁻¹ with the average number of rooting was 5.86 and the rooting rate was above 95.22%. The container seedlings can grow well and the survival rate was more than 90% when they were transplanted on the medium added with river sand and organic fertilizer with the ratio of 3∶1. The field experiments indicated that significant differences in increment and yield of pollen grains among the tissue-culture, cultivation and wild type of T. farfara under the same cultivation conditions. The cultivated plants were relatively high on the increment and yield of pollen grains. The active ingredient content of the tissue culture and the wild materials was basically the same.

Toxicity comparison between combination of Asteris Radix with Farfarae Flos and leaves of Tussilago farfara / 中草药

Objective: To compare the toxicity of two kinds of Ziwansan, which were prepared by Asteris Radix with Farfarae Flos (FF) and the leaves of Tussilago farfara (FL), respectively, The results will provide scientific basis for the utilization of leaves of T. farfarae. Methods: The FF and FL were in combination ratio of 1:1 with Asteris Radix, respectively, and given to mice at a dose of 40 g/kg for 14 d. The drug toxicology was evaluated by serum biochemical indicators and histopathological examination, as well as 1H-NMR based metabonomic approach. Results: The mice liver showed obvious damage as revealed by serum biochemical indicators and histopathological examination. Totally 15 biomarkers related to liver toxicity were determined by multivariate statistics and KEGG metabolic pathway analysis. By analyzing the distance between drug treated groups and blank group in scatter plots and the level changes of hepatoxicity related biomarkers, it was found that Ziwansan prepared by FF and FL showed different toxic effects on mice metabolome. However, there was no evidence that Ziwansan made from FL showed stronger toxicity than that made from FF. Conclusion: These results suggest that FL and FF show equivalent toxicity in Ziwansan, which lays the foundation for the utilization of leaves of T. farfara.

Effects of Tussilago farfara by Grinding Dispersion Treatment on the Dispersion Stability of Xiao’er Feike Granules in Water / 中国药房

OBJECTIVE:To improve the dispersion stability in water of Tussilago farfara powder,and to improve compliance of Xiao’er feike granules. METHODS:The effects of 4 kinds of dispersion stabilizer (sodium hexametaphosphate, dextrin, PEG4000 and lecithin) on dispersion stability of suspension in water were investigated during the grinding of T. farfara using rate of absorbance change(β)and Zeta potential as index;IR spectrum of samples were characterized. Using original formulation with-out dispersion stabilizer as control,the dispersion stability of new formulation granules in water were analyzed comparatively after adding dispersion stabilizer. RESULTS:Among 4 kinds of dispersion stabilizer,β of sample prepared by sodium hexametaphos-phate was the lowest,while Zeta potential of it was the highest;compared with original T. farfara,β of T. farfara grinded with 2.5% sodium hexametaphosphate decreased by 16.8%,and Zeta potential absolute value increased by 29.4%;no new peak was found in IR spectrum. Compared with control granules,granules suspension prepared by new formulation had lower β and higher Zeta potential absolute value (P<0.01);particle size was 30 μm and no large particle aggregation was found;β was less than 5.0% within 20 s sedimentation. CONCLUSIONS:During the preparation of Xiao’er feike granules,the application of sodium hexametaphosphate in the grinding of T. farfara powder can improve the dispersion stability of granules in water and the compliance of the preparation.

Optimization of ISSR-PCR reaction system of Tussilago farfara based on orthogonal design / 中国药学杂志

OBJECTIVE: To analyze the influence of main factors and second order interaction between main factors in Tussilago farfara ISSR-PCR systems and build ISSR reaction system with high stability and repeatability. METHODS: Based on the analysis of variance, an orthogonal table L27(313) was used to optimize the ISSR-PCR amplification system of T. farfara by four factors at three concentration levels, respectively. In addition, analysis of variance and range analysis were used to analyze the result of the experiment. RESULTS: A highly stable and repeatable ISSR-PCR reaction system was constructed. The factor Mg2+ had the most significant effect on T. farfara ISSR-PCR system (P<0.01), followed by Taq polymerase (P<0.05) and dNTPs (P<0.05). Among the second order interactions, the interaction of Mg2+ and dNTPs had the largest influence on the system (P<0.1). CONCLUSION: When building ISSR-PCR reaction system, special attention should be paid to the influence of the main factors and interaction between main factors on the whole ISSR-PCR system.