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.

A Comparative Analysis of the Anatomy, Phenolic Profile, and Antioxidant Capacity of Tussilago farfara L. Vegetative Organs.

Tussilago farfara L., a perennial species, is a medicinal herb used in traditional medicine, mainly for the treatment of respiratory tract-related pathology. In traditional Chinese medicine, flower buds are preferred; in Europe, the leaves are used; and in some parts of India, the whole plant is utilized. This preferential usage of the plant organs might be based on differences in the chemical composition due to environmental conditions, along with preferred traditional and cultural approaches. In this article, the impact of pedoclimatic growth conditions on the morpho-anatomical development and phytochemical profile of the plant were studied on T. farfara in the vegetative state, collected from two different locations in the Romanian spontaneous flora, revealing significant variations. Furthermore, the antioxidant profile of the specific extracts from the aerial and subterranean plant parts is also in accordance with these discrepancies. The plant anatomy was assessed histologically by optical microscopy, while the analytical chemistry evaluation was based on LC/MS and spectral methods for the evaluation of the antioxidant and enzyme inhibitory activity. To our knowledge, this is the first comparative analysis contextually reporting on the histology, phenolic profile, antioxidant capacity, and geographical location of the vegetative form of T. farfara.

The ethanol extract of flower buds of Tussilago farfara L. attenuates cigarette smoke-induced lung inflammation through regulating NLRP3 inflammasome, Nrf2, and NF-κB.

ETHNOPHARMACOLOGICAL RELEVANCE: The flower buds of Tussilago farfara L. (Abbreviated as FTF) were widely used in traditional Chinese medicine (TCM) to treat respiratory diseases, including asthma, dry throat, great thirst, turbid saliva, stinky pus, and coughs caused by various causes. AIM OF STUDY: The aim of study is to explore the efficiency of FTF in vitro and in vivo for the treatment of lung inflammation, and to illustrate the possible mechanisms of FTF in treating inflammation-related respiratory diseases targeting NOD-like receptor 3 (NLRP3) inflammasome, nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear transcription factor-κB (NF-κB). METHODS: Lung inflammation model in vivo was induced by exposure of mice to cigarette smoke (CS) for two weeks. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), inflammatory factors, and histology in lung tissues were investigated in presence or absence of ethanol extract of the flower buds of T. farfara L. (FTF-EtOH). In the cell-based models, nitric oxide (NO) assay, flow cytometry assay, enzyme-linked immunosorbent assay (Elisa), and glutathione (GSH) assay were used to explore the anti-inflammatory and anti-oxidant effects of FTF-EtOH. Possible anti-inflammatory mechanisms of FTF targeting NLRP3 inflammasome, Nrf2, and NF-κB have been determined using western blot, quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), immunofluorescence assay, nuclear and cytoplasmic extraction, and ubiqutination assay. RESULTS: FTF-EtOH suppressed CS-induced overproduction of inflammatory factors [e.g., tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß)], and upregulation of the content of intracellular MDA in the lung homogenate of mice. In cell-based models, FTF-EtOH reduced the lipopolysaccharide (LPS)-induced overproduction of inflammatory factors, and attenuated the CS extract-induced overgeneration of reactive oxygen species (ROS). Furthermore, FTF-EtOH up-regulated Nrf2 and its downstream genes through enhancing the stability of Nrf2 protein, and inhibited the activation of NF-κB and NLRP3 inflammasome, which have been confirmed by detecting the protein levels in the mouse model. CONCLUSIONS: FTF-EtOH effectively attenuated lung inflammation in vitro and in vivo. The protection of FTF-EtOH against inflammation was produced by activation of Nrf2 and inhibitions of NF-κB and NLRP3 inflammasome. These datas definitely support the ethnopharmacological use of FTF as an anti-inflammatory drug for treating respiratory diseases in TCM.

Biochemical Responses of Medicinal Plant Tussilago farfara L. to Elevated Heavy Metal Concentrations in Soils of Urban Areas.

This study was conducted in Tyumen (Russian Federation) to establish the effects of heavy metals' (Cu, Zn, Fe, Mn, Pb, and Cd) accumulation in soil and coltsfoot, as well as plants' biochemical responses to such an accumulation. The mobile and acid-soluble heavy metal fractions in soils, and the heavy metal contents in plants, were determined by atomic absorption spectrophotometry. The Cu, Zn, Fe, Mn, and Pb concentrations in soils exceeded background values. Pb content at the battery manufacturing plant was above the maximum permitted concentration. The percentages of the mobile heavy metal fractions decreased in the following order: Mn > Zn > Cu > Fe. The greatest heavy metal accumulation in soils and plants was found at the battery manufacturing and metallurgical plants examined in our study. Heavy metals' accumulation in the aboveground part of Tussilago farfara decreased in the following order: Fe > Zn > Cu > Mn > Pb > Cd. The accumulation of heavy metals stimulated the synthesis of photosynthetic pigments by 6-30%. Heavy metals provoked oxidative stress in cells, increasing the concentration of lipid peroxidation in products by up to 80%. Plant phenolics and flavonoids in the urban area of our study decreased compared to those in the control by 1.05, reaching up to 6.5 times. The change in coltsfoot catalase activity both increased and declined. Biochemical responses and heavy metal accumulation in coltsfoot from urban areas limit its use for medicinal purposes.

A review of the ethnobotanical value, phytochemistry, pharmacology, toxicity and quality control of Tussilago farfara L. (coltsfoot).

ETHNOPHARMACOLOGICAL RELEVANCE: Tussilago farfara L. (commonly called coltsfoot), known as a vital folk medicine, have long been used to treat various respiratory disorders and consumed as a vegetable in many parts of the world since ancient times. AIM OF THE REVIEW: This review aims to provide a critical evaluation of the current knowledge on the ethnobotanical value, phytochemistry, pharmacology, toxicity and quality control of coltsfoot, thus provide a basis for further investigations. MATERIALS AND METHODS: A detailed literature search was obtained using various online search engines (e.g. Google Scholar, Web of Science, Science Direct, Baidu Scholar, PubMed and CNKI). Additional information was sourced from ethnobotanical literature focusing on Chinese and European flora. The plant synonyms were validated by the database 'The Plant List' (www.theplantlist.org). RESULTS: Coltsfoot has diverse uses in local and traditional medicine, but similarities have been noticed, specifically for relieving inflammatory conditions, respiratory and infectious diseases in humans. Regarding its pharmacological activities, many traditional uses of coltsfoot are supported by modern in vitro or in vivo pharmacological studies such as anti-inflammatory activities, neuro-protective activity, anti-diabetic, anti-oxidant activity. Quantitative analysis (e.g. GC-MS, UHPLC-MRMHR) indicated the presence of a rich (>150) pool of chemicals, including sesquiterpenes, phenolic acids, flavonoids, chromones, pyrrolizidine alkaloids (PAs) and others from its leaves and buds. In addition, adverse events have resulted from a collection of the wrong plant which contains PAs that became the subject of public concern attributed to their highly toxic. CONCLUSIONS: So far, remarkable progress has been witnessed in phytochemistry and pharmacology of coltsfoot. Thus, some traditional uses have been well supported and clarified by modern pharmacological studies. Discovery of therapeutic natural products and novel structures in plants for future clinical and experimental studies are still a growing interest. Furthermore, well-designed studies in vitro particularly in vivo are required to establish links between the traditional uses and bioactivities, as well as ensure safety before clinical use. In addition, the good botanical identification of coltsfoot and content of morphologically close species is a precondition for quality supervision and control. Moreover, strict quality control measures are required in the studies investigating any aspect of the pharmacology and chemistry of coltsfoot.

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.

Trace metal uptake by native plants growing on a brownfield in France: zinc accumulation by Tussilago farfara L.

Several human activities such as mining, smelting, or transportations lead to trace metal pollution in soil. The presence of these pollutants can represent environmental and organism health risks. Phytoextraction can be used to remediate trace metal-contaminated soils. It uses the plants' ability to remove trace metals from soil and to accumulate them in their shoots, which can then be harvested. We studied the spontaneous vegetation growing on a brownfield located in France. The use of native plants is interesting since spontaneous vegetation is already well adapted to the site's environmental conditions leading to a better survival and growth than non-native plants. Ten native plant species were sampled, and the Cr, Cu, Cd, Ni, Pb, and Zn concentrations present in their shoots were measured. In order to determine the plant's capacity to extract trace metals from the soil, the bioconcentration factor (BCF) was calculated for each plant and trace metal. Plants with a BCF greater than 1 are able to accumulate trace metals in their shoots and could be a good candidate to be used in phytoextraction. Results underscored one new accumulator plant for Zn, Tussilago farfara L., with a BCF value of 3.069. No hyperaccumulator was found among the other sampled plants. Our preliminary study showed that T. farfara is able to accumulate zinc in its shoots. Moreover, this native plant is a pioneer species able to quickly colonize various habitats by vegetative multiplication. That is why T. farfara  L. could be interesting for zinc phytoextraction and could be worth further studies.

Chromane Enantiomers from the Flower Buds of Tussilago farfara L. and Assignments of Their Absolute Configurations.

Fourteen chromane derivatives of seven pairs of enantiomers (1-14) have been obtained from the ethanolic extract of the flower buds of Tussilago farfara L. Their structures with absolute configurations have been elucidated by detailed spectroscopic analyses, chemical methods, and particularly comparison of experimental ECD spectra with theoretically computed ones. Biological evaluations revealed that they did not show cytoprotective, antimicrobial, and α-glucosidase inhibitory activities.

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.

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.

Structural characterization of a polysaccharide from the flower buds of Tussilago farfara, and its effect on proliferation and apoptosis of A549 human non-small lung cancer cell line.

In present study, we purified a polysaccharide, TFPB1, from the flower buds of Tussilago farfara using DEAE-cellulose 52 anion-exchange and Sephacryl S-300 HR gel filtration chromatography. TFPB1 was a homogeneous polysaccharide with a molecular weight of 37.8kDa and composed of rhamnose, galacturonic acid, glucose, galactose, and arabinose, in a ratio of 13:13:1:7:12. Methylation and NMR results demonstrated that TFPB1 contained a rhamnogalacturonan I backbone consisting of a repeat disaccharide unit →4)-α-D-GalAp-(1→2)-α-L-Rhap-(1→, substituted by various type II arabinogalactan branches including terminal galactose, (1→3)-ß-D-galactan and (1→5)-α-L-arabinan, attached to the O-4 of (1→2)-α-L-Rhap. TFPB1 was found to inhibit cell proliferation of A549 cells and induce cell apoptosis in vitro. Furthermore, TFPB1 downregulated the phosphorylation of Akt, and upregulated caspase-3, Fas, FasL, and Bax expression, but downregulated Bcl-2 expression. Therefore, TFPB1 exhibited anti-proliferative and anti-apoptotic effect partly depending on the suppression of Akt signaling pathway. These findings provided us a potential chemotherapeutic strategy for the treatment of human non-small cell lung cancer.

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.

Preparation of Sesquiterpenoids from Tussilago farfara L. by High-speed Counter-current Chromatography.

BACKGROUND: Sesquiterpenoids, such as tussilagone, has effects of raising blood pressure, antiplatelet aggregation, and anti-inflammation activities, which is regarded as index compound for quality control of Tussilago farfara L. OBJECTIVE: This study was aimed to obtain an effective method for fast isolation of sesquiterpenoids from T. farfara L. by high-speed counter-current chromatography (HSCCC). MATERIALS AND METHODS: A solvent optimization method for HSCCC was presented, i.e., the separation factors of compounds after the K values of solvent system should be investigated. RESULTS: A ternary solvent system of n-hexane:methanol:water (5:8:2, v/v/v) was selected and applied for the HSCCC, and 56 mg of tussilagone (2) was isolated from T. farfara L., along with two other sesquiterpenoids 5.6 mg of 2,2-dimethyl-6-acetylchromanone (1) and 22 mg of 14-acetoxy-7 ß-(3'-ethyl cis-crotonoyloxy)-lα-(2'-methylbutyryloxy)-notonipetranone (3) by HSCCC with high purities. Their chemical structures were elucidated by liquid chromatography-mass spectrometry and nuclear magnetic resonance experiments. CONCLUSION: These results offered an efficient strategy for preparation of potentially health-relevant phytochemicals from T. farfara L., which might be used for further chemical research and pharmacological studies by preparative HSCCC. SUMMARY: The real separation efficiency has been verified by analytical HSCCC.A solvent optimization method for HSCCC was presented and applied to separate and prepare active compounds.A method for rapid and effective separation of target compound Tussilagone with high yield and purity from the flower buds of Tussilago farfara.Two other compounds 2,2-Dimethyl-6-acetylchromanone and 14-acetoxy-7ß-(3'-ethyl cis-crotonoyloxy) -lα- (2'-methylbutyryloxy). notonipetranone hasbeen obtained with high purities from flower buds of Tussilago farfara. Abbreviations used: HSCCC: High-Speed Counter-Current Chromatography; LC-MS: Liquid Chromatograph-Mass Spectrometer; NMR: Nuclear Magnetic Resonance; TCM: Traditional Chinese Medicine; HPLC: High Performance Liquid Chromatography; ESI-MS: Electrospray Ionization Mass Spectrometry; PE: petroleum ether.

Sensitive determination of pyrrolizidine alkaloids in Tussilago farfara L. by field-amplified, sample-stacking, sweeping micellar electrokinetic chromatography.

Pyrrolizidine alkaloids are the toxic components in Tussilago farfara L. Due to the lack of standard substances for quantitative analysis and traces of pyrrolizidine alkaloids in total alkaloids, the full quality control of Tussilago farfara L has been limited. In this study, we aimed to solve the difficulty of determination of pyrrolizidine alkaloids and identify more components in the total alkaloids. An on-line preconcentration method has been applied to improve determining sensitivity of pyrrolizidine alkaloids in Tussilago farfara L. in which included field-amplified sample stacking and sweeping in micellar electrokinetic capillary chromatography. The main parameters that affected separation and stacking efficiency were investigated in details. Under the optimal conditions, the sensitivity enhancement factors obtained by the developed method for the analytes were from 15- to 12-fold, the limits of detection of senkirkine and senecionine were 2∼5 µg/L. Senkirkine and senecionine have been detected in alkaloids (c) of Tussilago farfara L, along ferulic acid methyl ester and methyl caffeate. The developed method was also applied to the analysis of acid extraction (a) of Tussilago farfara L, and senkirkine could be detected directly. The results indicated that the developed method is feasible for the analysis of pyrrolizidine alkaloids in Tussilago farfara L with good recoveries.

Application of microscopy technique and high-performance liquid chromatography for quality assessment of the flower bud of Tussilago farfara L. (Kuandonghua).

BACKGROUND: Quality control is one of the bottleneck problems limiting the application and development of traditional Chinese medicine (TCM). In recent years, microscopy and high-performance liquid chromatography (HPLC) techniques have been frequently applied in the quality control of TCM. However, studies combining conventional microscopy and HPLC techniques for the quality control of the flower bud of Tussilago farfara L. (Kuandonghua) have not been reported. OBJECTIVE: This study was undertaken to evaluate the quality of the flower bud of T. farfara L. and to establish the relationships between the quantity of pollen grains and four main bioactive constituents: tussilagone, chlorogenic acid, rutin and isoquercitrin. MATERIALS AND METHODS: In this study, microscopic examination was used to quantify microscopic characteristics of the flower bud of T. farfara L., and the chemical components were determined by HPLC. The data were analyzed by Statistical Package for the Social Sciences statistics software. RESULTS: The results of the analysis showed that tussilagone, chlorogenic acid, rutin and isoquercitrin were significantly correlated with the quantity of pollen grains in the flower bud of T. farfara L. There is a positive correlation between them. From these results, it can be deduced that the flower bud of T. farfara L. with a greater quantity of pollen grains should be of better quality. CONCLUSION: The study showed that the established method can be helpful for evaluating the quality of the flower bud of T. farfara L. based on microscopic characteristic constants and chemical quantitation.