Erucic acid from Isatis indigotica Fort. suppresses influenza A virus replication and inflammation in vitro and in vivo through modulation of NF-kB and p38 MAPK pathway

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.

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.

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.

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.

Photosynthesis and metabolite responses of Isatis indigotica Fortune to elevated [CO2]

Climate change is affecting global crop productivity, food quality, and security. However,few studies have addressed the mechanism by which elevated CO_2 may affect the growth of medicinal plants. Isatis indigotica Fortune is a widely used Chinese medicinal herb with multiple pharmacological properties. To investigate the physiological mechanism of I.indigotica response to elevated [CO_2], plants were grown at either ambient [CO_2](385 μmol mol^(-1)) or elevated [CO_2] (590 μmol mol^(-1)) in an open-top chamber (OTC)experimental facility in North China. A significant reduction in transpiration rate (T_r) and stomatal conductance (g_s) and a large increase in water-use efficiency contributed to an increase in net photosynthetic rate (Pn) under elevated [CO_2] 76 days after sowing. Leaf non-photochemical quenching (NPQ) was decreased, so that more energy was used in effective quantum yield of PSII photochemistry (Φ_(PSⅡ)) under elevated [CO_2]. High ΦPSII,meaning high electron transfer efficiency, also increased Pn. The [CO_2]-induced increase in photosynthesis significantly increased biomass by 36.8%. Amounts of metabolic compounds involved in sucrose metabolism, pyrimidine metabolism, flavonoid biosynthesis, and other processes in leaves were reduced under elevated [CO_2]. These results showed that the fertilization effect of elevated [CO_2] is conducive to increasing dry weight but not secondary metabolism in I. indigotica.

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.

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

陇东地区菘蓝干物质和品质动态积累研究及评价

目的探讨陇东地区菘蓝干物质和药用成分积累规律,并确定其最佳采收期。方法通过定期调查菘蓝农艺指标和HPLC法测定板蓝根中尿苷、鸟苷、(R,S)-告依春、腺苷含量积累变化规律,应用灰色关联度法评价不同时期板蓝根的综合利用价值。结果株高、株幅、叶长、叶宽、叶片数、叶重、根长、根粗、根重,以及尿苷、鸟苷、(R,S)-告依春、腺苷含量等指标在不同生长时期存在显著变化,并且各指标的峰值不在同一时期;不同品系间上述各指标也存在明显差异。隆德菘蓝和2008-6-2综合利用价值的加权关联度在10月9日最高,2008-11和2016-2在10月24日最高,整体上2008-6-2和2016-2种质优于2008-11和隆德菘蓝。结论建议陇东地区菘蓝最佳采收期为10月中下旬。

药用植物菘蓝的遗传及远缘杂交研究进展

十字花科菘蓝为我国大宗常用中药材植物,其叶和根分别是大青叶和板蓝根的原料。本文总结了菘蓝的基因组结构、生物活性成分及其生物合成途径、远缘杂交及新材料创建等方面的研究进展。菘蓝(2n=2x=14)的基因组大小为300 Mb,为tPCK核型,3万余蛋白编码基因。高质量的基因组测序解析了菘蓝的主要生物活性成分吲哚生物碱、苯丙烷类、萜类的合成途径及其候选基因。由于染色体消除,菘蓝(父本)与白菜及甘蓝型油菜的族间有性杂交只产生了具有少数菘蓝遗传成分的非预期杂种。菘蓝与萝卜的体细胞杂种具有双亲染色体,但自交及回交均未产生后代。菘蓝与白菜的体细胞杂种具有加倍的菘蓝染色体组,花粉部分可育而雌性不育。菘蓝与甘蓝型油菜的体细胞杂种与甘蓝型油菜连续回交后创建了全套的7个甘蓝型油菜-菘蓝附加系,一些附加系具有比菘蓝更强的广谱抗病毒效果;体细胞杂交中发生的双亲线粒体基因组重组导致甘蓝型油菜新细胞质雄性不育系的产生,菘蓝特定染色体上的育性基因导入培育了恢复系。最后讨论了这些附加系对菘蓝遗传研究的价值及开发利用前景。

菘蓝碳氮代谢与基质C/N对氮处理的动态响应

为研究施氮后基质C/N和菘蓝(Isatis indigotica Fort.)碳氮代谢的动态变化,以河北居群的菘蓝为试验材料,播种后施用0、5、15、20 g·L^(-1)CO(NH_(2))_(2),分别记为CK、N1、N2、N3。利用重铬酸钾外加热法和碳氮分析仪测定基质及菘蓝碳、氮元素含量;实时荧光定量PCR(qRT-PCR)法测定菘蓝碳氮代谢酶基因的表达;超高效液相色谱(UPLC)法测定菘蓝指标成分含量。结果表明,菘蓝碳氮代谢对不同浓度氮营养的响应不同。随着处理时间延长,N1、N2、N3叶与根的可溶性糖含量先增加后减少,与磷酸烯醇式丙酮酸羧化酶(PEPC)基因表达量变化趋势相反,与蔗糖磷酸合成酶(SPS)活性变化趋势相似。菘蓝碳氮代谢产物与碳氮代谢关键酶的活性及基因表达量密切相关。N1靛蓝含量与硝酸还原酶(NR)基因表达量变化趋势相似,靛玉红含量与谷氨酰胺合成酶(GS)基因表达量、谷氨酸合成酶(GOGAT)活性变化趋势相似,与GOGAT基因表达量、GS活性变化趋势相反。N2靛蓝含量与NR活性变化趋势相反,与GOGAT基因表达量变化趋势相似,靛玉红含量与NR、GS基因表达量、GOGAT活性变化趋势相似。N3靛蓝、靛玉红含量与NR、GS基因表达量、GOGAT活性变化趋势相似。GS、GOGAT活性和其相应酶基因表达量的变化趋势相反。随着菘蓝的生长,基质中的碳元素含量逐渐增加,氮元素含量逐渐减少,C/N升高。菘蓝碳、氮元素含量的变化趋势与基质相反。施氮后,菘蓝叶、根中累积的氮素含量增多,基质中的氮素减少,碳元素含量的变化与氮相反。N2处理各种酶的活性及基因表达量较高,同时累积更高的活性成分。综上,15 g·L^(-1)CO(NH_(2))_(2)处理更有助于菘蓝指标成分的累积。本研究结果为菘蓝氮营养高效利用提供了理论参考。

基于板蓝根及其混伪品叶绿体基因组的mini-barcode开发及其定性能力的研究

[目的]基于板蓝根及其混伪品的叶绿体基因组序列开发mini-barcode并验证其鉴定能力,为板蓝根的质量控制和科学评价提供方法。[方法]从GenBank数据库中下载板蓝根及其混伪品所有已发表的叶绿体基因组序列,导入PhyloSuite v1.2.1软件提取其共有蛋白编码基因并分别对齐,后续使用DnaSP 6软件进行核苷酸多态性分析以筛选出候选DNA mini-barcode区域。通过Geneious软件多序列比对,筛选高变区用于mini-barcode开发。针对候选条形码区域设计引物。采用试剂盒法提取板蓝根药材总DNA分别利用通用ITS2引物和该实验设计引物进行聚合酶链式反应(PCR)和测序。[结果]基于叶绿体基因组的核苷酸多态性分析结果,选择accD基因作为候选mini-barcode开发区域并设计了扩增引物。通过遗传距离和测序数据分析,该分子标记能被成功扩增并实现鉴定。[结论]开发了用于鉴定板蓝根及其混伪品的mini-barcode,为实现DNA降解的板蓝根药材及含板蓝根的中药制剂等混合样本的真伪鉴别奠定基础。

光强对菘蓝生长、化学成分及抗氧化活性的影响

【目的】探究光强对大青叶基原植物菘蓝Isatis indigotica生长、药材大青叶化学成分及抗氧化活性的影响,为大青叶基原植物菘蓝的人工栽培提供参考。【方法】在100%全光照、60%全光照、20%全光照下种植菘蓝,观测菘蓝生长旺盛期叶片生长情况,同时测定大青叶靛蓝、靛玉红、总黄酮、总多糖、总游离氨基酸等化学成分质量分数和抗氧化活性。【结果】菘蓝叶片生长与光强呈正相关,100%全光照组的菘蓝株高、叶长、叶宽、叶片数、叶面积均为最高;大青叶靛蓝、总黄酮、总多糖质量分数均随着光强的降低而降低,且各组间差异显著(P<0.05);靛玉红质量分数随光强的增加呈先升高后降低的趋势,60%全光照组显著高于其他处理(P<0.05),但100%全光照组与20%全光照组间差异不显著;总游离氨基酸质量分数随着光强的降低而升高。抗氧化活性与光强呈正相关,20%全光照组的1,1-二苯基-2-三硝基苯肼(DPPH)自由基半清除率(IC50)显著高于60%全光照组和100%全光照组(P<0.05)。【结论】100%全光照下大青叶药材产量最高,且内在品质最好,较60%和20%全光照更适合栽培。

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.

板蓝根苯丙素糖苷成分研究

为研究板蓝根中苯丙素糖苷类化学成分,采用大孔吸附树脂D101、正、反相硅胶柱色谱以及半制备型高效液相色谱等技术分离纯化其乙醇提取物,结合多种现代波谱技术及量子化学计算鉴定化合物的结构,从板蓝根乙醇提取物中分离出13个苯丙素糖苷类化合物,其结构分别鉴定为isatisoside A(1),ceplignan-4-O-β-D-glucoside(2),isatioxyneolignoside E(3),isatioxyneolignoside F(4),lariciresinol-4'-O-β-D-glucopyranoside(5),lariciresinol-9-O-β-D-glucopyranoside(6),conoicaoside(7),medioresinol di-O-β-D-glucopyranoside(8),顺紫丁香苷(9),紫丁香苷(10),(Z)-coniferin(11),coniferin(12),1-feruloyl-O-β-D-glucopyranoside(13)。化合物2,7,8为首次从板蓝根中分离得到,化合物1为新化合物,命名为isatisoside A。

板蓝根提取物对仔猪生长性能、血液生化指标及免疫机能影响

文章旨在探究不同水平的板蓝根提取物对断奶仔猪生长性能、血液生化指标及免疫机能影响。试验将80头健康的体重(20±0.5)kg“杜×长×大”三元杂交断奶仔猪随机分为4组,每组20个重复,每个重复1头仔猪,在基础日粮中添加0(1组)、0.5%(2组)、1.0%(3组)、1.5%(4组)板蓝根提取物,预试验为10 d,试验期为60 d。结果表明:(1)试验3、4组平均日增重较1组比分别显著提高11.6%、10.1%(P<0.05),其料重比较1组比分别显著降低9.2%、7.3%(P<0.05);(2)试验3、4组血清中TP、GLB含量较1组分别显著提高16.4%、16.8%、21.3%、26.9%(P<0.05),其血清中AST、BUN含量较1组分别显著降低20.5%、22.0%、13.3%、11.3%(P<0.05)。(3)试验3、4组血清中IgA、IgG、IL-2含量较1组分别提高13.6%、12.7%、11.6%、12.5%、29.0%、21.9%(P<0.05),其血清中TNF-α含量较1组分别降低20.5%、21.8%(P<0.05)。综上所述,不同水平的板蓝根提取物可以提高仔猪的生长性能,改善血液生化水平,提高免疫机能,板蓝根提取物以1.0%的添加比例较适宜。

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.

不同添加剂对板蓝根茎叶发酵品质与营养价值的影响

为研究纤维素酶、复合乳酸菌等添加剂对板蓝根茎叶发酵品质和营养价值的影响,试验设置为8个处理组,分别为纤维素酶组(BX)、复合乳酸菌组(BR)、青干贮专用复合酶组(ZB)、青贮宝组(BQ)、青贮宝+纤维素酶组(BQX组)、纤维素酶+复合乳酸菌组(BRX组)、青干贮专用复合酶+复合乳酸菌组(BRZ组)、对照组(CK组),每组3个重复,试验期60 d,对比分析各组发酵饲料营养品质和发酵品质。结果显示:各试验处理的板蓝根茎叶均无腐败臭味呈淡酸味,褐黄色,质地松散不粘手,评价为良好,与对照组相比,BX、ZB、BQX组气味得分分别提高了23.33%、16.67%、30.00%(P<0.05)。BX、ZB、BQX、BRX组乳酸含量分别提高了10.43%、12.70%、14.13%、13.05%(P<0.01);BRX、BQX组丙酸含量分别降低了59.09%、68.18%(P<0.01);BQX组有机酸总和提高了16.44%(P<0.01);BRX组乳酸占比提高了4.37%(P<0.01);与对照组相比,BX、BQX、BRX组粗纤维含量分别降低了24.78%、31.62%、30.15%(P<0.01)。添加纤维素酶、复合乳酸菌、纤维素酶+复合乳酸菌酶菌共用等,可有效改善板蓝根茎叶发酵品质,但对于其营养价值未有显著改善,同时未检出(R,S)-告依春等活性成分。

遗传算法在河西绿洲菘蓝灌溉决策优化中的应用

为缓解河西绿洲菘蓝种植水资源供需矛盾,解决灌溉过程中存在的灌溉决策粗放问题,提高灌溉水利用效率,参照Jensen模型、水量平衡方程等,根据河西绿洲干旱少雨的环境特征及菘蓝种植过程中各生育期耗水强度不同的生长规律,构建了菘蓝灌溉决策模型并利用遗传算法对有限水资源在菘蓝灌溉中的合理分配进行求解。同时综合考虑各生育期灌溉水量约束条件、有效降水量、灌溉成本和产量等因素,在非充分灌溉理论指导下,利用遗传算法结合现有菘蓝种植数据对灌溉决策进行优化,以达到节水、高效、高产的目的。优化结果显示,肉质根轻度水分亏缺可以显著提高灌溉水利用效率,同实际种植灌溉情况相符。

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)

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.