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Isatis indigotica Fort. (Ban-Lan-Gen) is an herbal medicine prescribed for influenza treatment. However, its active components and mode of action remain mostly unknown. In the present study, erucic acid was isolated from Isatis indigotica Fort., and subsequently its underlying mechanism against influenza A virus (IAV) infection was investigated in vitro and in vivo. Our results demonstrated that erucic acid exhibited broad-spectrum antiviral activity against IAV resulting from reduction of viral polymerase transcription activity. Erucic acid was found to exert inhibitory effects on IAV or viral (v) RNA-induced pro-inflam-matory mediators as well as interferons (IFNs). The molecular mechanism by which erucic acid with antiviral and anti-inflammatory properties was attributed to inactivation of NF-kB and p38 MAPK signaling. Furthermore, the NF-kB and p38 MAPK inhibitory effect of erucic acid led to diminishing the transcriptional activity of interferon-stimulated gene factor 3 (ISGF-3), and thereby reducing IAV-triggered pro-inflammatory response amplification in IFN-β-sensitized cells. Additionally, IAV- or vRNA-triggered apoptosis of alveolar epithelial A549 cells was prevented by erucic acid. In vivo, erucic acid administration consistently displayed decreased lung viral load and viral antigens expression. Meanwhile, erucic acid markedly reduced CD8+cytotoxic T lymphocyte (CTL) recruitment, pro-apoptotic signaling, hyperactivity of multiple signaling pathways, and exacerbated immune inflammation in the lung, which resulted in decreased lung injury and mortality in mice with a mouse-adapted A/FM/1/47-MA(H1N1) strain infection. Our findings provided a mechanistic basis for the action of erucic acid against IAV-mediated inflammation and injury, suggesting that erucic acid may have a therapeutic potential in the treatment of influenza.
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Objective: To study the glycosides from the roots of Isatis indigotica. Methods: The chemical components were isolated and purified by silica gel, Sephadex LH-20, ODS C18 gel, and semi preparative-HPLC chromatographic techniques while the structures were deduced by NMR, MS and IR spectral data analysis. Results: A total of 12 glycosides were obtained and elucidated as isatindigoside C (1), epiprogoitrin (2), progoitrin (3), o-aminobenzoic acid 7-O-β-D-glucopyranosyl ester (4), 2’-O-methyladenosine (5), 2-methoxyphenyl β-D-glucopyranoside (6), coniferin (7), syringin (8), cis-coniferin (9), cis-syringin (10), corchoionoside C (11), and cannabiside D (12). Conclusion: Compound 1 is a new indole alkaloid glycoside which named as isatindigoside C, while compounds 5-7 and 9-12 are isolated from the plant for the first time.
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Objective Identifying laccases, as one of the key synthetases in the lariciresinol biosynthetic pathway, by analyzing the transcriptome sequencing results in Isatis indigotica would provide a dependable foundation for later functional study of Isatis indigotica′s laccases. Methods Bioinformatical softwares and kinds of analytical methods online were used to find out the characteristics of the laccases from I. indigotica, including physical and chemical properties, homology, and the properties after induction of MeJA. Results The transcriptional results showed that Iilac3 and Iilac5 from I. indigotica were corresponded ith the accumulation of the effective metabolites, making them the potential functional genes participated in lariciresinol synthesis. Conclusion Through the detailed bioinformatical analysis of Iilacs,which laid a solid foundation for the further study of the physiological and biochemical mechanisms and structural characteristics of the functional proteins.
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Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21 (DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene (IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that IiPAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of IiPALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I. indigotica.
Subject(s)
Amino Acid Sequence , Base Sequence , Cloning, Molecular , Gene Expression Regulation, Plant , Isatis , Genetics , Molecular Sequence Data , Multigene Family , Phenylalanine Ammonia-Lyase , Chemistry , Genetics , Metabolism , Phylogeny , Plant Proteins , Chemistry , Genetics , Metabolism , Sequence AlignmentABSTRACT
Isatidis Radix,the root of Isatis indigotica Fort.(Cruciferae),is a representitive herb widely used for clearing away heat-toxin in traditional Chinese medicine (TCM).Lariciresinol is a representitive component of lignans and an important efficacious substance with the antiviral effect.This review elucidated the progress on its biosynthetic pathways,the screening of key regulatory genes and metabolic engineering of lignans components in Isatidis Radix,providing a favorable reference for the full understanding of biosynthesis of antiviral active components,the quality improvement of Isatidis Radix and the sustainable utilization of TCM resources.
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Objective: To clone cinnamate 4-hydroxylase (C4H) gene from Isatis indigotica and to analyze its bioinformatics and expression. Methods: The full-length cDNA of C4H was 1 674 bp (GenBank accession No. GU014562) long with an open reading frame (ORF) of 1 530 bp encoding a polypeptide of 509 amino acid residues. The comparison of C4H genomic DNA sequences and C4H cDNA sequence revealed that the C4H genomic DNA contained two introns. Southern-blotting analysis indicated that C4H was a multiple copy gene. C4H expression could be detected in all tissues at different expression levels, with the strongest expression in the roots. Further expression analysis revealed that the signaling components of defense/stress pathways, such as ultraviolet-B radiation (UV-B), methyl jasmonate (MeJA), abscisic acid (ABA), and Gibberellins (GA3) could up-regulate the C4H transcript levels compared with the control. Conclusion: We have first extracted the full length cDNA of C4H gene from I. indigotica, and its structural and bioinformatic analyses are carried out, which will help us to further illuminate this pathway. The research also provides a possibility to study the antiviral active constiuents in I. indigotica by plant secondary metabolic engineering.
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Objective: To investigate the physical location of 45S, 5S rDNA, and telomere sequences and analyze the karyotype of the metaphase chromosomes of Isatis indigotica. Methods: Multicolor fluorescence in situ hybridization (FISH) approach was used to detect the physical location of the above sequences. Results: One pair of 45S rDNA sites located on chromosome 4, one pair of 5S rDNA sites located on chromosome 5 and 14 pairs of telomere sites located at the ends of each chromosome in I. indigotica were detected. The karyotype formula is 2n = 2x = 14 = 10m (2SAT) + 4sm, which belongs to 2A karyotype. Conclusion: This research provides effective cytological markers for karyotype analysis and construction of a molecular cytogenetic map of I. indigotica.
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OBJECTIVE:To observe the ultra-filtration and the resistance to pollution of5ultra-filtrated membrane in respect of each of the compositions from the Isatis indigotica Fort.water-extraction,in order to produce the Isatis indigotica Fort.-preparation through ultra-filtrated method instead of ethanol-sedimentary method.METHODS:By measuring the quantities of the solid residue and the contents of polysaccharide,arginine and chloroform extraction in the ethanol-sedi?mentary solution and ultra-filtrated solution by gravimetry,TLC-scan and HPLC respectively.RESULTS:The solid residue in the ultra-filtered solution was lower than that in the ethanol-sedimentary solution;The content of polysaccharide in the ultra-filtered solution with polypropylene nitrile membrane and polysulfone-6000membrane were close to that in the ethanol-sedimentary solution.The arginine in the ultra-filtered solution was higher than that in the ethanol-sedimentary solution,but the chloroform extraction in the ultra-filtered solution was lower than that in ethanol-sedimentary solution;The resistance to pollution of polysulfone membrane was the strongest in these ultra-filtrated membrane.CONCLUSION:According to the requirement for isolation of active components,ultra-filtrated method with polysulfone-50000membrane could be used instead of ethanol-sedimentary method for efficient and economical application.
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Object To study chemical constituents of the root of Isatis indigotica Fort Methods The powdered plant material was percolated with 95% ethanol, the percolate was extracted with different solvents, the extract was subject to chromatography on silica gel column and macroporous resin column. The compounds were identitfied by their physicochemical properties and spectral data (MS, 1HNMR, 13 CNMR, UV and IR) Results Two compounds were obtained from the ethanol extracts of the plant root They are 3 (2′ hydroxyphenyl) 4(3H) quinazolinone and isaindigodione respectively Conclusion The two compounds were obtained from I. indigotica for the first time
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Objective Two insect resistance genes Bacillus thuringiensis(Bt) crystal protein gene CryIA(c) and cowpea trypsin inhibitor gene CpTI were introduced into tetraploid Isatis indigotica to enhance the resistance to moths.Methods I.indigotica was transformed with a plasmid,pGBI121S4ABC,containing CryIA(c) Bt and CpTI and the selectable gene(Npt-Ⅱ) driven by the CaMV35S promoter via Agrobacterium tumefaciens LBA4404 mediated transformation.Then PCR and Southern blotting assay were conducted followed by moth bioassay test.Results Co-transgenic rate of the two genes in tetraploid I.indigotica was 16.67%.The integration and expression of introduced genes in T0 regenerated transgenic plants were confirmed by Southern blotting assays.Insect bioassay test demonstrated transgenic lines had significant inhibition to moths,compared with wild type control.Conclusion Co-transformation of Bt and CpTI genes is an effective strategy to enhance the resistance to moths for tetraploid I.indigotica.
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Object To study the chemical constituents in the ro ot of Isatis indigotica Fort Methods The s eparations were carried out by column chromatography and identified by advanced physical and spectral data analysis Results Five compounds were isolated and identified as neohesperidin (Ⅰ), ammonium formate (Ⅱ), isol iquiritigenin (Ⅲ), liquiritigenin (Ⅳ), and adenosine (Ⅴ) Conclusio n Neohesperidin, liquiritigenin and isoliquiritigenin are isolated f o r the first time from the plants of Cruciferae, ammonium formate is obtained fro m the root of I indigotica for the first time
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Object To extract and separate the chemical constituents from the root of Isatis indigotica Fort (Cruciferae) Methods The root of I. indigotica was percolated with 95% ethyl alcohol, partitioned in solvents of different polarities and finally isolated on silica gel and macroporous resin columns The purified compounds obtained were identified and structurally elucidated by their physicochemical properties and spectral analysis Results Two compounds were obtained and named as isaindigotidione (Ⅰ) and (E)-3-(3′, 5′-dimethoxy-4′-hydroxybenzylidene)-2-indolinone Conclusion The two compounds were new
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Objective To study the growth differences of Isatis indigotica sown in spring and autmn,reproductive identity and seedy condition of I.indigotica sown in various times,so as to provide the theoretical foundation for scientific standardization of cultivating process.Methods I.indigotica was sampled at a fixed period.At each stage,the growth of seedling and root system was recorded,blooming and seedy condition was recorded in efflorescence.Results The results showed that I.indigotica turned into genesial cycle after thermostage and the change of plant height and crown of I.indigotica were evidence.There was obvious differences between I.indigotica sown in different times.I.indigotica sown in spring grew better than that sown in autumn before efflorescence,but it grew worse than I.indigotica sown in autumn after seeding.Conclusion The weight of thousand seeds,average yield of single plant,and acre yield are different between the two ways for reserving seeds,but the difference of germination rate of seed is indistinctive,it will not influence the yield next year.The seed quality of I.indigotica sown in spring is better than that sown in autumn,but it must pay regard to the plant falling problem.The two ways are both feasible in the south of China while sown in spring is more suitable in the north of China.
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AIM: To study the best harvest time of Isatis Indigotica Fort.. METHODS: The adenosine content was determined by HPLC, and at the same time, the dry substance accumulation, the alcohol extract of the herb were also compared at the different growth periods of Isatis indigotica Fort.. RESULTS:During its whole growth period, the contents of adenosine and alcohol extract increase and very fast before November. The dry substance accumulates highly in December. CONCLUSION: In consideration of the yield and the contents of adenosine and alcohol extrat, the best harvest time should be between November and December.