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.

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.

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。

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.

引发处理对不同温度下菘蓝种子萌发与幼苗生长的生态响应

以菘蓝种子为试材,采用培养皿发芽的方法,研究了不同温度下不同质量分数PEG 6000对菘蓝种子萌发及幼苗生长的影响,以期为促进菘蓝在非适宜条件下的生长提供参考依据。结果表明:引发剂处理对菘蓝种子发芽率、发芽势、发芽指数、幼苗高度、相对电导率均有所影响。PEG 6000引发可提高菘蓝种子萌发率,并在同一温度下,随着PEG 6000浓度增大,发芽率降低,但均高于未用引发剂引发的对照组(CK);随着温度升高,种子萌发期缩短。在低温(10℃)下使用引发剂后,种子发芽势、相对电导率与对照组之间存在显著差异。通过试验可以推测,使用PEG 6000引发剂可以预防菘蓝苗期的冻害,且最佳质量分数为10%,有利于菘蓝出苗整齐、长势健壮,提高幼苗的抗逆能力。

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。

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。

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。

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)

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 。

板蓝根化学成分的研究(Ⅲ)

目的 提取分离板蓝根化学成分。方法 板蓝根用 95 %乙醇渗漉 ,不同极性溶剂分级萃取 ,再分别用硅胶和大孔树脂作柱层析分离 ,测定纯化合物的理化常数和波谱数据 ,鉴定化学结构。结果 从板蓝根中分得 2个化合物 ,分别命名为 3 -羟苯基喹唑酮〔3 - ( 2′- hydroxypheny1) - 4 ( 3 H) - quinazolinone〕,依靛蓝酮 ( isaindigodione)。结论 该

大青叶和板蓝根提取物的抑菌作用研究

目的 :探讨中药大青叶和板兰根的逐级提取物的抑菌作用。方法 :采用试管稀释法测定大青叶和板兰根的各级提取物—总浸液、乙醇提取液、正丁醇萃取液对各实验菌的最小抑菌浓度 (MIC)。结果 :各级提取物对实验菌分别有不同程度的抑菌作用 ,其中对金黄色葡萄球菌的抑菌作用最为明显 ;各提取物的抑菌强度依次为 :正丁醇萃取液 (对三种实验菌均明显抑制 :MIC≤ 62mg/ml)、乙醇提取液 (金黄色葡萄球菌 :MIC≤ 62mg/ml、肠炎杆菌 :MIC≤ 1 2 5mg/ml、大肠埃希菌MIC≤ 50 0mg/ml)、总浸液 (仅对金黄色葡萄球菌抑制明显 :MIC =62mg/ml)。结论 :大青叶和板蓝根具有广谱抗菌作用 。

水分胁迫对菘蓝生长发育和有效成分积累的影响

目的：研究不同水分对菘蓝生长发育和主要有效成分的影响。方法：采用室外盆栽水分胁迫试验方法,对水分胁迫条件下菘蓝需水规律、叶绿素含量、蒸腾速率、光合速率、生物量指标以及有效成分靛玉红积累进行了研究。结果：水分胁迫程度增加,需水量、叶绿素含量降低,蒸腾速率、光合作用和生物量均降低,但水分利用效率提高。结论：菘蓝需水的关键时期是7月,严重水分胁迫和水分过多都不利于靛玉红含量积累,中度水分胁迫能促进根部靛玉红含量积累,大青叶7月为最佳采收期,板蓝根10月采收较适宜。当田间最大持水量为45%~70%,菘蓝的产量与质量可以兼优。

逆境胁迫对菘蓝幼苗靛玉红含量的影响

培养30d的菘蓝无菌幼苗经250mmol／L NaCl、自然干旱、20mmol／L ABA、4℃低温等不同方式逆境处理，不同时间内取样，用高效液相色谱法对其中靛玉红的含量进行测定。结果表明：盐、干旱、ABA处理12h内能提高菘蓝幼苗中靛玉红含量59.05％～161．67％，低温胁迫处理24h也能提高靛玉红含量141．4％，说明适当逆境胁迫处理能有效提高菘蓝幼苗体内靛玉红的累积量，其中250mmol／L NaCl胁迫处理效果最为显著。

板兰根中三种拟除虫菊酯杀虫剂的残留分析

研究了板蓝根中3种拟除虫菊酯杀虫剂残留分析方法。以石油醚为萃取溶剂，分别用索氏抽 提法、浸渍振荡法、超声波法提取。提取液经100 mL丙酮∶0.05 mol/L氯化钙溶液(1∶1)液 -液分配净化后，以弗罗里硅土柱净化，5%乙酸乙酯-石油醚淋洗，气相色谱法电子捕获检 测器测定。结果表明，方法的线性范围为0.4×10-10～4×10-10g，氯氰菊酯 、氰戊菊酯、溴氰菊酯最低检测浓度分别为5×10-3、5×10-3、2×10-3 mg/kg。3种提取方法对板蓝根的添加回收率、变异系数符合农药残留分析的要求。

不同氮素形态及其比例对菘蓝生物学特性的影响

采用砂培法栽培菘蓝,以菘蓝叶与根的干重、主根长度与直径、叶绿素含量和光合参数为指标,研究三种氮素形态,即硝态氮(NO-3-N)、铵态氮(NH+4-N)和酰胺态氮[CO(NH2)2-N]四种不同浓度对菘蓝生物学性质的影响,为菘蓝栽培生产中高效利用氮肥提供理论依据。结果表明,氮素处理能增加菘蓝叶与根的干重,铵态氮对菘蓝主根长度及侧根数的影响最显著,而酰胺态氮与硝态氮对主根直径的影响相近,硝态氮对菘蓝的叶干重、根干重与根冠比的影响最为显著;不同形态氮素处理能显著提高叶绿素含量与净光合速率,硝态氮对叶绿素和净光合速率有显著影响,酰胺态氮对气孔导度与蒸腾速率的影响最显著,铵态氮对叶片的胞间二氧化碳浓度有显著影响。不同形态氮素通过对菘蓝叶片的叶绿素含量和光合参数的影响实现对其生长状况的影响,其中硝态氮对菘蓝生长的影响最为显著。

RP-HPLC法测定板蓝根药材中核苷类成分的含量

目的:建立了中药材板蓝根中尿苷、腺苷和鸟苷的反相高效液相色谱含量测定方法。方法:采用 Agilent ZORBAX SB-C_(18)分析柱(250mm×4.6mm,5μm),流动相为10mmoL·L^(-1)磷酸二氢钠(1%磷酸调 pH 2.9)-甲醇(3:97);柱温25℃;流速0.8mL·min^(-1);检测波长254nm。结果:尿苷、腺苷和鸟苷的线性范围分别为10.7～107.0,10.1～101.1,10.6～106.0μg·mL^(-1);相关系数分别为0.9996,0.9996,0.9995。平均回收率(n=3)分别为97.2%,101.6%,97.3%;RSD 分别为1.1%,3.3%,1.2%。结论:采用反相高效液相色谱法同时测定板蓝根中尿苷、腺苷和鸟苷3种成分的含量,方法简便、快速、准确,结果可靠。

板蓝根化学成分研究

目的 分离鉴定板蓝根中的化学成分。方法 用硅胶、ODS等柱色谱分离纯化 ,以核磁、质谱、紫外、红外光谱及理化常数鉴定结构。结果 分离鉴定了 5个化合物 ,为新橙皮苷 ( )、甲酸铵 ( )、异甘草素 ( )、甘草素 ( )和腺苷 ( )。结论 其中新橙皮苷、甘草素和异甘草素为首次从十字花科中分离得到 ;甲酸铵为首次从该植物中分离得到。