Effects of Different Exogenous Substances on Seed Germination of Isatis indigotica Under Drought Stress and Chemical Composition of Isatis indigotica leaves

This study was to investigate the effects of three exogenous substances on chemical constituents of Isatis indigotica leavesand their efficacy in alleviating drought stress, and explore the methods of applying exogenous substances to efficient cultivationof Isatis indigotica. Polyethylene glycol (PEG) was used to simulate drought stress to deal with seeds of Isatis indigotica at thegermination stage (concentration: 0, 10%, 15%, and 20%). Simultaneous operation of exogenous growth regulators [microbialinoculum (MI), γ-aminobutyric acid (GABA) and salicylic acid (SA)] and PEG were implemented in seeds of Isatis indigotica.The effects of drought stress and the mitigation of exogenous substances were observed by statistics of seed germination potential,germination rate, hypocotyl length, and radicle length of each treatment. The effects of exogenous substances on the content ofalkaloids, crude protein and free amino acids in the leaves of Isatis indigotica grown in a greenhouse were determined after sprayingexogenous substances on the plants. The differences of germination potential, germination rate, hypocotyl length, and radicle lengthamong 15% PEG stress treatment, 10% PEG stress treatment and the control were significant (P<0.05). According to the predesignedgermination standard, the seeds did not germinate under 20% PEG stress treatment. When the PEG concentration was 15%, the resultsof seed germination potential and germination rate after adding MI were significantly different from those under stress alone (P<0.05).When exposed to 10% PEG stress, the supplementation of GABA led to a notable increase in radicle length of Isatis indigotica seeds,showing significant differences compared to other three treatments. The application of MI and GABA under 15% PEG stress resultedin a significant increase in the radicle and hypocotyl length of Isatis indigotica seeds compared to other two treatments. The contentof the total alkaloids in leaves of Isatis indigotica was significantly increased after spraying GABA. Meanwhile, the contents of crudeprotein and the total free amino acids were kept constant after spraying exogenous substances. Application of MI and GABA couldalleviate drought stress of Isatis indigotica. The content of the total alkaloids in leaves of Isatis indigotica could significantly increaseafter spraying GABA.

A New Alkaloid from the Root of Isatis indigotica Fort

A E-2-[(3'-indole)cyanomethylene]-3-indolinone was isolated from the root of Isatis indigotica Fort. The structure elucidation and 1H, 13C NMR assignments were achieved by spectroscopic method.

Alkaloids from Isatis indigotica

Isaindigotone 1 and 3-(2'-hydroxyphenyl)-4(3H)-quinazolinone 2 were isolated from the roots of Isatis indigotica. Compound 1 was elucidated as a new alkaloid.Compound 2 was discovered for the first time from a natural source

Isolation and characterization of lignans from the leaves of Isatis indigotica Fortune

A phytochemical investigation of 80%EtOH exact of Isatis indigotica Fortune leaves yielded ten lignans(1-10),including six furofurans lignans(1-6),one arylnaphthalenes lignans(7)and three tetrahydrofurans lignans(8-10).Their structures were determined by HRESIMS spectroscopic data and extensive NMR analyses.Among them,compounds 6,9 and 10 were isolated from the genus oi Isatis for the first time.

Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica

Objective AP2/ERF(APETALA2/ethylene-responsive factor)superfamily is one of the largest gene families in plants and has been reported to participate in various biological processes,such as the regulation of biosynthesis of active lignan.However,few studies have investigated the genome-wide role of the AP2/ERF superfamily in Isatis indigotica.This study establishes a complete picture of the AP2/ERF superfamily in I.indigotica and contributes valuable information for further functional characterization of IiAP2/ERF genes and supports further metabolic engineering.Methods To identify the IiAP2/ERF superfamily genes,the AP2/ERF sequences from Arabidopsis thaliana and Brassica rapa were used as query sequences in the basic local alignment search tool.Bioinformatic analyses were conducted to investigate the protein structure,motif composition,chromosome location,phylogenetic relationship,and interaction network of the IiAP2/ERF superfamily genes.The accuracy of omics data was verified by quantitative polymerase chain reaction and heatmap analyses.Results One hundred and twenty-six putative IiAP2/ERF genes in total were identified from the I.indigotica genome database in this study.By sequence alignment and phylogenetic analysis,the IiAP2/ERF genes were classified into 5 groups including AP2,ERF,DREB(dehydration-responsive element-binding factor),Soloist and RAV(related to abscisic acid insensitive 3/viviparous 1)subfamilies.Among which,122 members were unevenly distributed across seven chromosomes.Sequence alignment showed that I.indigotica and A.thaliana had 30 pairs of orthologous genes,and we constructed their interaction network.The comprehensive analysis of gene expression pattern in different tissues suggested that these genes may play a significant role in organ growth and development of I.indigotica.Members that may regulate lignan biosynthesis in roots were also preliminarily identified.Ribonucleic acid sequencing analysis revealed that the expression of 76 IiAP2/ERF genes were up-or down-regulated under salt or drought treatment,among which,33 IiAP2/ERF genes were regulated by both stresses.Conclusion This study undertook a genome-wide characterization of the AP2/ERF superfamily in I.indigotica,providing valuable information for further functional characterization of IiAP2/ERF genes and discovery of genetic targets for metabolic engineering.

Cloning and Characterization of a Differentially Expressed Phenylalanine Ammonialyase Gene （liPAL） After Genome Duplication from Tetraploid Isatis indigotica Fort.

Phenylpropanoid derivatives are a complex class of secondary metabolites that have many important roles in plants during normal growth and in responses to environmental stress. Phenylalanine ammonialyase （PAL） catalyzes the first step in the biosynthesis of phenylpropanoids. In the present study, we isolated a novel phenylalanine ammonialyase gene （designated as liPAL） from tetraploid Isatis indigotica Fort. by rapid amplification of cDNA ends （RACE）, which was a cultivar from the diploid plant by genome duplication. The full-length cDNA of liPAL was 2 530-bp long with an open reading frame （ORF） of 2 178 bp encoding a polypeptide of 725 amino acid residues. Analysis of liPAL genomic DNA revealed that it was structurally similar to other plant PAL genes, with a single intron at a conserved position, and a long highly conserved second exon. Semi-quantitative RT-PCR revealed that the liPAL expression in roots and leaves from a tetraploid sample was higher than that in diploid progenitor, whereas expression of liPAL in stems was almost the same as each other. Furthermore, the highest expression of liPAL in tetraploid plant was found in roots, which was found in stems in diploid plants. Further expression analysis revealed that gibberelUn （GA3）, abscisic acid （ABA）, methyl jasmonate （MeJA） and cold treatments could up-regulate the liPAL transcription in tetraploid plants. All our findings suggest that liPAL participates not only in the defense/stress responsive pathways, but also probably in the polyploidy evolution of L indigotica.

Effect of Microwave and He-Ne Laser on Enzyme Activity and Biophoton Emission of Isatis indigotica Fort

Abstract: The seed embryos of Isatis indigotica Fort were exposed to He-Ne laser (5.23 mW/mm2, radiated for 5 min) and microwave (1.26 mW/mm2, radiated for 8 s) irradiation to determine the effects of microwave and He-Ne laser pretreatment on enzyme activities, and biophoton emission of cotyledon. Then: (i) changes in the activities of enzymes in I. indigotica cotyledon (such as amylase, transaminase, and proteinase) were measured to investigate the effects of He-Ne laser and microwave pretreatment; and (ii) biophoton emission was measured to determine the speed of cell division and metabolism. Results from these experiments indicated that: (i) the activities of amylase, transaminase, and proteinase of the cotyledon pretreated by He-Ne laser and microwave were significantly increased; and (ii) the intensity of biophoton emission was enhanced significantly by He-Ne laser and microwave irradiation. These changes suggest that He-Ne laser and microwave pretreatment can improve the inner energy of seeds, lead to an enhancement of cotyledon enzymes, and speed up the metabolism of the cell, resulting in significantly increased biophoton emission. Moreover, the mechanism of action of the effects of laser and microwave radiation on the microcalorimetric parameters, enzyme activities, and biophoton emission of seeds is discussed on the basis of the results obtained.

Indole alkaloid sulfonic acids from an aqueous extract of Isatis indigotica roots and their antiviral activity

Six new indole alkaloid sulfonic acids(1–6), together with two analogues(7 and 8) that were previously reported as synthetic products, were isolated from an aqueous extract of the Isatis indigotica root. Their structures including the absolute configurations were determined by spectroscopic data analysis, combined with enzyme hydrolysis and comparison of experimental circular dichroism and calculated electronic circular dichroism spectra. In the preliminary assay, compounds 2 and 4 showed antiviral activity against Coxsackie virus B3 and influenza virus A/Hanfang/359/95(H3N2), respectively.

8,4′-Oxyneolignane glucosides from an aqueous extract of“ban lan gen”(Isatis indigotica root)and their absolute configurations

Three pairs of glycosidic 8,4′-oxyneolignane diastereoisomers, named isatioxyneolignosides A-F(1–6), were isolated from an aqueous extract of Isatis indigotica roots. Their structures and absolute configurations were elucidated by comprehensive spectroscopic data analysis and enzyme hydrolysis. The validity of Δδ_(C8-C7) values to distinguish threo and erythro aryl glycerol units and Cotton effects at 235±5 nm to determine absolute configurations at C-8 in 1–6 and their aglycones(1a–6a) are discussed.

The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.:molecular cloning,characterization,and expression analysis

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 Ii PAL1 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 Ii PALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I.indigotica.

Two pairs of unusual scalemic enantiomers from Isatis indigotica leaves

Two pairs of unusual scalemic enantiomers with a dimethoxyphenyl）ethyl]-2-methoxyindolin-3-one, novel carbon skeleton of 2-[1′-（4＂-hydroxy-3＂,5＂- named isatidifoliumindolinones A-D （1-4）, were isolated from an aqueous extract of lsatis indigotica leaves （da qing ye）. Their structures including absolute configurations were determined by spectroscopic data analysis combined with comparison of their experimental CD and calculated ECD spectra. Validity of the ECD spectra calculation to assign the absolute configurations is discussed. Plausible biosynthetic pathways of 1-4 are proposed. Stereochemistry-dependent activity against LPS-induced NO production in BY2 cells was observed, and among the stereoisomers compound 4 is most active.

Isatindolignanoside A, a glucosidic indole-lignan conjugate from an aqueous extract of the Isatis indigotica roots

A glucosidic indole-lignan conjugate with a novel carbon skeleton, named isatindolignanoside A（1）, was isolated from an aqueous extract of the Isatis indigotica roots ＂ban lan gen＂. Its structure was elucidated by comprehensive analysis of spectroscopic data, including J-and NOESY correlation-based configurational analysis, circular dichroism（CD） data, enzyme hydrolysis, and theoretical ECD calculation. In a preliminary assay, compound 1 showed antiviral activity against Coxsackie virus B3. This compound is the first example of natural product having a structural feature of conjugation between indole and lignan units, and its biosynthetic pathway is postulated.

Indole alkaloid glycosides with a 1′-(phenyl)ethyl unit from Isatis indigotica leaves

Seven indole alkaloid glycosides containing a 1′-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl unit(1-7)were isolated from an aqueous extract of Isatis indigotica leaves(da qing ye).Their structures were determined by spectroscopic data analysis combined with enzymatic hydrolysis as well as comparison of their experimental CD(circular dichroism)and calculated ECD(electrostatic circular dichroism)spectra.Based on analysis of[α]D20 and/or Cotton effect(CE)data of 1-7,two simple roles to assign location and/or configuration ofβ-glycopyranosyloxy and 1′-(phenyl)ethyl units in the indole alkaloid glycosides are proposed.Stereoselectivity in plausible biosynthetic pathways of 1-7 is discussed.Compounds 3 and 4 and their mixture in a 3:2 ratio showed activity against KCNQ2 in CHO cells.The mixture of 5 and 6(3:2)exhibited antiviral activity against influenza virus H1 N1 PR8 with IC5064.7μmol/L(ribavirin,IC5054.3μmol/L),however,the individual 5 or 6 was inactive.Preliminary structure-activity relationships were observed.

Isatis indigotica:from(ethno)botany,biochemistry to synthetic biology

Isatis indigotica Fort.(Chinese woad)is a species with an ancient and well-documented history as an indigo dye and medicinal plant.It is often confused with Isatis tinctoria L.(European woad),a medicinal plant in Europe.Here,the differences between I.indigotica and I.tinctoria are systematically described.The usage development history,clinical applications and pharmacological activities,and chemical components of I.indigotica are also summarized.Lignans,indole alkaloids,and their corresponding derivatives have been identified as the major active ingredients of I.indigotica and are associated with anti-viral,anti-inflammatory,anti-cancer,and other health-promoting activities.Notable progress has been made in understanding the biosynthetic pathway and regulation mechanism of lignans and indole alkaloids in I.indigotica,the results from which should facilitate the process of targeted metabolic engineering or synthetic biology.Moreover,multiple biotechnology methods such as polyploid breeding and genetic engineering have been used with I.indigotica to result in,for example,greater yields,higher levels of bioactive component accumulation,and enhanced stress tolerance to salt,drought,and insects.Some issues require additional analyses,and suggestions for future research on I.indigotica are also discussed.

Discovery of alkaloids from the leaves of Isatis indigotica Fortune with neuroprotective activity

Seven alkaloids including five undescribed ones(1 a/1 b,2,3 and 5)were obtained from the leaves of Isatis indigotica Fortune.Their structures were established by extensive spectroscopic analyses.The absolute configurations of compounds 1 a,1 b,3 and 5 were determined by comparison of the experimental and calculated electronic circular dichroism(ECD)spectra.Subsequently,the neuroprotective effects of all the isolates against H_(2)O_(2)-induced injury in SH-SY5 Y cells were evaluated in vitro by MTT assay.Moreover,Annexin V-FITC/PI double staining was performed,while the activities of antioxidant enzymes(SOD,CAT and GSH-Px)for compounds 1 a and 1 b were measured.

QUALITY-DESIGN IN TRADITIONAL CHINESE MEDICINE: A CASE STUDY ABOUT ISATIS INDIGOTICA FORT

Quality is the basis for the efficacy of Traditional Chinese Medicine(TCM),affecting herbs,formulations,and even the practice of TCM itself.In our laboratory,we used Isatis indigotica,a prevalent Chinese medicinal herb,as a model to illustrate strategies and methods for TCM Quality-design study.First of all,tetraploid I.indigotica(2n=28)with better yield,higher antiviral activity and enhanced resistance was obtained from its natural diploid progenitor(2n=14)

菘蓝转录因子IiMYB的基因克隆及表达特征

MYB转录因子在植物生长发育、次生代谢产物生物合成以及抗逆应激等多个方面发挥重要作用。为了研究菘蓝中特异MYB转录因子的表达特征,本研究采用cDNA末端快速扩增(RACE)技术克隆菘蓝中目标MYB转录因子编码基因IiMYB的全长c DNA并获得其基因组DNA序列,通过RT-qPCR检测IiMYB在四倍体菘蓝和二倍体菘蓝各器官以及不同胁迫条件下的表达情况。IiMYB的c DNA序列全长926 bp(GenBank登录号:DQ468346),含600 bp的开放阅读框(ORF),编码199个氨基酸,其对应的基因组DNA序列含有3个外显子和2个内含子。生物信息学分析表明IiMYB与拟南芥的AtMYBL2同源性最高,遗传距离最近,并仅含有一个myb域(R3)。IiMYB在两种倍性菘蓝的根茎叶中均有表达,其中叶中最高,且在四倍体中的表达水平均高于二倍体。胁迫因素高盐(NaCl)、茉莉酸甲酯(MeJA)、赤霉素(GA3)、脱落酸(ABA)和水杨酸(SA)处理均能上调IiMYB的表达水平,而低温(4℃)处理对IiMYB的表达水平无显著影响。本研究首次从菘蓝植物中克隆得到MYB转录因子并进行了表达分析,为进一步阐明与MYB转录因子相关的四倍体菘蓝优良性状形成的分子机制提供一定基础,也为通过分子育种培育优质菘蓝品系提供了条件。

Aromatic compounds from an aqueous extract of “ban lan gen” and their antiviral activities

A pair of new diphenyl glycerol ether enantiomers(-)-l and(+)-l and two new methyl benzamidobenzoates 2 and 3,named(-)-(R)-and(+)-(S)-isatindigotrioic acid[(-)-l and(+)-l]and isatindigoticamides A(2) and B(3),respectively,were isolated from an aqueous decoction of the roots of Isatis indigotica(ban lan gen).Their structures were elucidated by spectroscopic data analysis including2 D NMR experiments.The absolute configurations of(-)-l and(+)-l were assigned based on the CD exciton chirality method.Compounds 2 and 3 exhibited antiviral activities against HSV-1 with IC_(50)values of 4.87 and 25.87μmol/L,respectively.Compound 2 was also found active against Coxsackie virus B3 and LPS-induced NO production.