A Comparative Study on Microstructure between Isatis indigotica Fort.and Baphicacanthus cusia(Nees) Bremek.

[Objective] This study aimed to compare the microstructure between I. in- digotica Fort. and B. cusia （Nees） Bremek. [Method] Microstructure of I. indigotica Fort. and B. cusia （Nees） Bremek. with different sources were compared and analyzed by using digital microscopy. [Result] Comparison result of the microstructure shows that reticulate vessels, single starch granules, multiple starch granules and stone cells are mostly observed in I. indigotica Fort.; pitted vessels, parenchyma cells with small pits, single starch granules and cystolith are mostly observed in B. cusia （Nees） Bremek., which indicated that microstructure of L indigotica Fort. and B. cusia （Nees） Bremek. are significantly different. [Conclusion] Microstructure char- acteristics of I. indigotica Fort. and B. cusia （Nees） Bremek. established in this study can be used to evaluate the quality of these two kinds of Chinese herbal medicines, which also provide scientific basis and methods for comprehensively studying the quality standard of /. indigotica Fort. and B. cusia （Nees） Bremek. Key words Isatis indigotica Fort.; Baphicacanthus cusia （Nees） Bremek.; Microstruc- ture; Quality

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.

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.

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

喷施木醋液对菘蓝苗期生长和生理的影响

以小叶菘蓝为试材,采用温室盆栽法,研究了清水浸种和15%PEG 6000浸种胁迫下,叶面喷施不同浓度木醋液对菘蓝幼苗生长和生理指标的影响,以期为木醋液应用于菘蓝及其他药用植物的绿色、生态种植提供参考依据。结果表明:叶面喷施适宜浓度的木醋液显著促进菘蓝幼苗生长,缓解种子受PEG 6000胁迫对幼苗生长的抑制作用,提高叶绿素含量、光合速率、蒸腾速率、气孔导度和水分利用率,降低丙二醛(MDA)含量及超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性,以400倍液效果最显著。

菘蓝叶绿体基因组密码子使用偏好性分析

为探究菘蓝(Isatis indigotica Fort.)叶绿体基因组密码子使用偏好性及其影响因素,本研究使用Codon W 1.4.2软件和在线软件CUSP对筛选到的52个蛋白质编码序列进行密码子使用模式分析。结果表明:菘蓝叶绿体基因组密码子总GC含量(Total GC content of codon,GC_(all))为37.59%,密码子第1位碱基GC含量(GC content of the first base of codon,GC_(1))>密码子第2位碱基GC含量(GC content of the second base of codon,GC_(2))>密码子第3位碱基GC含量(GC content of the third base of codon,GC_(3)),且均小于50%,说明密码子第3位碱基拥有较低的GC含量且密码子偏好使用A或U。有效密码子数(Effective number of codon,ENC)值为35.92~55.32,密码子适应指数(Codon adaptation index,CAI)值为0.10~0.31,密码子偏好指数(Codon bias index,CBI)值为-0.24~0.20,最优密码子使用频率(Frequency of optimal codons,FOP)值为0.24~0.54,说明密码子使用偏好性较弱;通过中性绘图分析、PR2-plot分析、ENC-plot分析和对应性分析,发现菘蓝叶绿体基因组密码子使用偏好性受选择压力和突变等因素的共同影响,主要因素为自然选择。最优密码子分析共筛选出14个最优密码子,且均以A或U结尾。以上研究可为菘蓝的系统进化、环境适应性和种质改良提供理论基础。

4种根际促生菌对菘蓝生长与生理的影响

[目的]本文旨在研究4种不同根际促生菌对菘蓝根际环境改变、生长及生理的影响,为菘蓝绿色生产提供理论参考。[方法]采用4种根际促生菌(肠杆菌GS-22、芽胞杆菌GS-27、鞘氨醇杆菌GS-72和不动杆菌GS-103)单施的方法开展盆栽试验,测定土壤理化指标、菘蓝生长指标、光合指标、营养物质与相关酶活性以及活性成分等,评价根际促生菌对菘蓝的促生效果。[结果]4种根际促生菌对土壤理化性质有不同程度影响,GS-22和GS-72能够提高土壤全氮含量,GS-22和GS-103显著提高土壤速效磷含量,各处理土壤有机质含量均显著提高。接种根际促生菌,土壤脲酶、土壤碱性磷酸酶和土壤蔗糖酶活性较不接种土壤根际菌(CK)的增幅分别为33.78%~73.70%、49.51%~83.93%和8.10%~186.26%。4种根际促生菌促进菘蓝生长,增加株高、主根直径、干重、鲜重等,其中GS-22对菘蓝生长性状影响最显著。根际促生菌对菘蓝生理也有显著影响,各处理菘蓝硝酸还原酶、蔗糖合成酶、酸性磷酸酶活性较CK分别增加53.81%~125.19%、57.64%~76.54%和7.87%~22.78%。4种根际菌有利于菘蓝营养物质(可溶性糖、游离氨基酸)与活性成分(总黄酮、靛蓝、靛玉红)的积累,其中GS-22与GS-27处理均可提高总黄酮、靛蓝和靛玉红的含量。[结论]菘蓝根际促生菌GS-22、GS-27、GS-72和GS-103具促生能力,能够改良土壤环境,对菘蓝的生长与生理过程有促进作用,其中GS-27的综合效果最佳。所筛选的菌株可为开发菘蓝专用微生物菌肥、实现药材增产增效提供策略。

河西灌区板蓝根氮磷钾养分需求规律研究

通过对不同氮磷钾施用水平下板蓝根的生长动态和产量及药用成分的测定,得出河西灌区板蓝根的氮磷钾养分需求量,以指导河西灌区板蓝根生产的科学施肥。以当地板蓝根主栽品种安徽亳州种为试材,开展了板蓝根氮磷钾养分需求规律研究。结果表明,河西灌区板蓝根生产中N的适宜用量为255~300 kg/hm^(2),苗期、生长旺盛期、收获期对N的需求量分别占整个生育期需氮量的9.21%、43.65%、47.14%;P_(2)O_(5)的适宜用量为150~225 kg/hm^(2),苗期、生长旺盛期、收获期对P_(2)O_(5)的需求量分别占整个生育期P_(2)O_(5)需量的15.80%、25.91%、58.29%;K_(2)O的适宜用量为150~225 kg/hm^(2),苗期、生长旺盛期、收获期对K_(2)O的需求量分别占整个生育期K_(2)O需量的20.83%、19.38%、59.79%。

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.

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。

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)

低磷胁迫对菘蓝幼苗叶片生理特性的影响

为研究低磷胁迫对菘蓝幼苗叶片的影响,本研究将菘蓝种子种于以蛭石为基质的小花盆中,用含不同浓度(0 mmol/L、0.05 mmol/L、0.5 mmol/L)KH_(2)PO_(4)的霍格兰营养液处理13 d,培养条件为温度25℃、光照强度80μmol/(m^(2)·s)、相对湿度为75%、光照时间为12 h光照/12 h黑暗,观察生理特性的变化。结果显示,KH_(2)PO_(4)浓度为0.5 mmol/L时,菘蓝幼苗生长状态最好,叶片长度可达4.5 cm;KH_(2)PO_(4)浓度低于0.5 mmol/L时,随着KH_(2)PO_(4)浓度的降低(0.05 mmol/L、0 mmol/L),菘蓝幼苗叶片的生理特性显著变化,与对照相比,呼吸速率、净光合速率、气孔导度、胞间二氧化碳浓度、总叶绿素含量和含水量分别下降了53.8%和30.8%、61.3%和38%、72.3%和33.1%、45.1%和27.2%、38.3%和13.3%、6.7%和3.7%;总可溶性糖含量、脯氨酸含量、电导率、丙二醛(MDA)含量、过氧化氢含量和超氧阴离子含量分别上升了41.6%和32.4%、54.9%和24.4%、58.7%和19.6%、151%和59.8%、94.1%和23.5%、78.9%和47.4%;超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)、单脱氢抗坏血酸还原酶(MDHAR)、脱氢抗坏血酸还原酶(DHAR)和谷胱甘肽还原酶(GR)活性分别上升了17.2%和10.9%、57.8%和42.2%、59%和15.7%、32.5%和27%、24.6%和5.7%、36.1%和22.7%、45.5%和33.2%。表明低磷处理能抑制菘蓝幼苗叶片的光合作用,并对叶片造成渗透胁迫,菘蓝幼苗叶片可能通过合成渗透保护物质调节细胞渗透压,并通过抗氧化酶清除活性氧。

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.

高效液相色谱法测定板蓝根中主要游离氨基酸的含量

目的:采用柱前衍生法测定板蓝根中5种游离氨基酸含量。方法:采用异硫氰酸苯酯柱前衍生,高效液相色谱法测定了板蓝根中精氨酸、苏氨酸、丙氨酸、脯氨酸和缬氨酸的含量。色谱柱:Phenomenex ODS 3(5μm,250 mm×4.6 mm);流动相A:醋酸钠缓冲溶液-乙腈(370:28),流动相B:乙腈-水(4:1),流速1 mL·min^(-1),流动相A与B比例,0.00 min为100:0;18.00 min为91:9;18.01 min为80:20;40.00 min为67:33;40.01 min为0:100,保持5.00 min。柱温43℃,测定波长254 nm;结果:本方法测定精氨酸、苏氨酸、丙氨酸、脯氨酸和缬氨酸的线性范围分别为0.016-0.47(r=0.9993),0.0018—0.072(r=0.9995),0.0022—0.086(r=0.9994),0.016-0.80(r=0.9997),0.0019—0.097 mg·mL^(-1)(r=0.9998);平均回收率分别为94.9%,108.8%,98.2%,99.0%和96.6%。结论:本法不需要专门的氨基酸分析仪,操作简便,灵敏度高,结果准确可靠。

高效液相色谱指纹图谱应用于板蓝根的鉴定

利用高效液相色谱建立了板蓝根的色谱指纹图谱。采用反相C1 8柱 ,流动相为水∶甲醇 (V V =96∶4 ) ;检测波长为 2 6 0nm ;流速 1 0mL min进行试验。根据相对保留值α和相对面积Sr 对色谱指纹图谱进行分析对比研究 ,建立了一个快速、简易鉴别板蓝根样品的色谱指纹图谱分析方法。该法为中药样品的鉴定提供了较全面的信息 。