水杨酸诱发的NO介导了丹参悬浮培养细胞中丹酚酸B的生物合成

水杨酸(SA)可诱导丹参悬浮培养细胞中一氧化氮(NO)产生、苯丙氨酸解氨酶(PAL)活化及丹酚酸B(Sal B)的生物合成。为了阐明NO对丹参悬浮培养细胞中Sal B生物合成的影响及作用机理,本实验利用NO供体硝普钠(SNP)、NO合成酶抑制剂L-NNA(Nω-nitro-L-arginine)、NO淬灭剂c PITO(carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide)以及PAL抑制剂L-AOPP(L-2-aminooxygen-3-phenyl acrylic acid)分别处理丹参悬浮培养细胞,并对其胞内NO水平、PAL活性和Sal B积累量进行了检测。结果表明,硝普钠(SNP)处理显著促进了NO产生、PAL活性和Sal B的积累,而L-NNA和c PITO抑制上述过程,说明NO诱发PAL活性提高并参与了SA诱导的Sal B生物合成;L-AOPP显著抑制了PAL活性及Sal B积累,却对NO产生没有显著影响,揭示NO位于PAL的上游。这说明SA诱发的NO产生、PAL活化及Sal B合成之间存在因果关系,即NO通过激活PAL触发Sal B生物合成。     

绿豆苯丙氨酸解氨酶的性质及抗肿瘤作用研究

本文参照Ｈａｖｉｒ的方法,从绿豆中分离纯化了苯丙氨酸解氨酶（ＰＡＬ）。纯化的ＰＡＬ经ＳＤＳ聚丙烯酰胺凝胶电泳及等电聚焦电泳鉴定为单一的蛋白质区带,并测得亚基分子量为７６ＫＤ,等电点为５．４５。在ＰＡＬ对Ｌ１２１０小鼠淋巴细胞白血病细胞株的体外抑制实验观察到：ＰＡＬ对该瘤株的抑制作用随作用时间的延长和药物剂量的增加而增强,０．２Ｕ／ｍｌ、１．０Ｕ／ｍｌ、２．０Ｕ／ｍｌ、４．０Ｕ／ｍｌ、６．０Ｕ／ｍｌ、１０．０Ｕ／ｍｌ的ＰＡＬ作用癌细胞７２ｈ,其抑制率分别为２５．８％、４０．０％、５５．３％、７２．６％、７７．９％、８２．９％。     

NO对银杏悬浮细胞生长及黄酮类物质合成的影响

以硝普钠(sodium nitroprusside,SNP)为一氧化氮(NO)的供体,向银杏悬浮细胞培养液中加入不同浓度的SNP,研究外源NO对银杏悬浮细胞生长状况、过氧化氢酶(CAT)活性、苯丙氨酸解氨酶(PAL)活性和黄酮类物质生物合成的影响.结果表明,低浓度SNP有利于银杏悬浮细胞生长,而高浓度SNP可以促进黄酮类物质的合成.银杏悬浮细胞在添加0.5和10 mmol/L SNP的培养基中培养16 d时,细胞干重分别为对照组的134%和73%;在添加10 mmol/L SNP的培养基中培养20 d时,细胞中黄酮类物质的含量为对照组的136%.同时,10 mmol/L SNP促进银杏悬浮细胞PAL和CAT活性显著升高.NO专一性淬灭剂c-PITO(carboxyl phenyltetramethylimidazoleoxide)抑制SNP对银杏悬浮细胞生长、CAT活性、PAL活性和黄酮类物质含量的促进作用,说明SNP是通过其分解产物NO影响细胞生长和黄酮类物质的合成.根据这些结果推测,NO可能通过触发银杏悬浮细胞的防卫反应,激活了细胞中黄酮类物质的生物合成途径.     

干旱胁迫下外源一氧化氮促进银杏可溶性糖、脯氨酸和次生代谢产物合成

以硝普钠（sodium nitroprusside,SNP）为一氧化氮（NO）供体,研究其在干旱胁迫下对银杏叶片物质代谢的影响。结果表明,在土壤相对含水量为35％的干旱胁迫下,250μmol／LSNP显著提高可漆性糖含量、脯氨酸含量、苯丙氨酸解氨酶（phenyl Manineammonia lyase．PAL）活性、黄酮类化合物和银杏内酯含量。NO清除剂血红蛋白（hemoglobin）抑制了SNP对银杏叶片可溶性糖、脯氨酸、PAL酶活性、黄酮和银杏内酯合成的促进作用,说明SNP是通过其分解产物NO影响这些物质的合成。推测在干旱胁迫下次生代谢产物黄酮类物质和银杏内酯的作用类似脯氨酸和可溶性糖,NO通过促进这些物质的合成,来缓解干旱胁迫的不良影响。     

胡桐悬浮培养细胞中苯丙氨酸解氨酶活性与红厚壳素产量的关系

胡桐CR2细胞悬浮培养过程中,苯丙氨酸解氨酶(PAL)活性上升后红厚壳素产量即增加.加入真菌诱导子后,PAL活性与红厚壳素产量呈一定的正相关.以PAL抑制物降低PAL活性后,红厚壳素产量也降低;诱导物与抑制物同时加入,PAL活性与红厚壳素产量均界于诱导物处理与未处理之间.     

NO和H_2O_2在介导热激诱发金丝桃细胞合成金丝桃素中的信号互作

热激处理(40℃,10min)可以诱发金丝桃细胞中金丝桃素的生物合成并诱导细胞产生一氧化氮(NO)和过氧化氢(H2O2).过氧化氢酶(CAT)和NO专一性淬灭剂(cPTIO)不仅可以分别抑制由热激诱发的H2O2积累和NO合成,而且还可以阻断热激处理对金丝桃素生物合成的促进作用.H2O2单独处理虽然不能提高细胞的金丝桃素产量,但是H2O2和NO共同处理对金丝桃素产量的促进作用显著高于NO单独处理,表明NO和H2O2对金丝桃素的生物合成具有协同诱导效应.NO处理可以提高细胞的H2O2水平,而外源H2O2对金丝桃细胞的NO合成积累也具有促进作用,说明NO和H2O2对彼此的合成反应具有促进作用.CAT在抑制热激诱发H2O2合成的同时还能够部分抑制热激细胞中NO的合成,而cPITO也可以同时降低热激细胞的H2O2水平.上述实验结果提示,在热激处理下金丝桃细胞中的NO和H2O2可能通过互作反应提高各自的信号水平.质膜NAD(P)H氧化酶抑制剂DPI和NO合酶抑制剂PBITU可以抑制NO和H2O2之间的互作反应,并且解除NO和H2O2对金丝桃素合成的协同诱导作用,说明NO和H2O2对金丝桃素合成积累的协同效应依赖于两种信号分子之间的互作反应.本文实验结果不仅证实了NO和H2O2是参与热激诱发金丝桃细胞中金丝桃素合成所必需的两种信号分子,而且揭示了NO和H2O2在介导热激诱发金丝桃素生物合成过程中特殊的信号互作现象.     

NO和H2O2在介导热激诱发金丝桃细胞合成金丝桃素中的信号互作

热激处理（40℃,10min）可以诱发金丝桃细胞中金丝桃素的生物合成并诱导细胞产生一氧化氮（NO）和过氧化氢（H2O2）．过氧化氢酶（CAT）和NO专一性淬灭剂（cPTIO）不仅可以分别抑制由热激诱发的H2O2积累和NO合成,而且还可以阻断热激处理对金丝桃素生物合成的促进作用．H2O2单独处理虽然不能提高细胞的金丝桃素产量,但是H2O2和NO共同处理对金丝桃素产量的促进作用显著高于NO单独处理,表明NO和H2O2对金丝桃素的生物合成具有协同诱导效应．NO处理可以提高细胞的H2O2水平,而外源H2O2对金丝桃细胞的NO合成积累也具有促进作用,说明NO和H2O2对彼此的合成反应具有促进作用．CAT在抑制热激诱发H2O2合成的同时还能够部分抑制热激细胞中NO的合成,而cPITO也可以同时降低热激细胞的H2O2水平．上述实验结果提示,在热激处理下金丝桃细胞中的NO和H2O2可能通过互作反应提高各自的信号水平．质膜NAD（P）H氧化酶抑制剂DPI和NO合酶抑制剂PBITU可以抑制NO和H2O2之间的互作反应,并且解除NO和H2O2对金丝桃素合成的协同诱导作用,说明NO和H2O2对金丝桃素合成积累的协同效应依赖于两种信号分子之间的互作反应．本文实验结果不仅证实了NO和H2O2是参与热激诱发金丝桃细胞中金丝桃素合成所必需的两种信号分子,而且揭示了NO和H2O2在介导热激诱发金丝桃素生物合成过程中特殊的信号互作现象．     

独一味苯丙氨酸解氨酶基因的克隆与表达分析

以独一味叶片为材料,采用RT-PCR和RACE方法克隆了独一味苯丙氨酸解氨酶基因（PAL）的全长cDNA,命名为LrPAL基因。测序结果表明,LrPA L基因全长2 298 bp,含有1个2 145 bp的完整开放阅读框（ORF）,编码714个氨基酸。蛋白序列分析表明,其包含典型的PAL活性中心序列（GTITASGDLVPLSYIA）,与其他植物的PAL蛋白有很高的同源性。系统进化树分析表明,独一味LrPAL与唇形科植物的PAL蛋白聚为一类,说明两者的亲缘关系较近。用 Real-Time PCR方法检测发现,LrPAL基因在独一味的叶中表达量最高,茎中表达量最少。研究结果推测,从独一味中克隆获得的苯丙氨酸解氨酶基因（LrPAL）是典型的PAL家族成员,在独一味各组织发育过程中具有重要功能。     

茉莉酸甲酯(MeJA)对贯叶连翘悬浮细胞生长和贯叶金丝桃素产量的影响

培养基中添加100 μm01·L-1MeJA后,贯叶连翘悬浮细胞生物量增加量和贯叶金丝桃素(HF)的产量分别是未经MeJA处理的1.3倍和1.73倍;培养3 d时,培养基中添加100 μmol·L-1 MeJA能提高HF产量,是未经MeJA处理的2.4倍.     

广西金丝桃属植物有效成分金丝桃素的含量测定研究

采用 UV-VIS法和 HPLC法检测了广西产金丝桃属植物及其提取物中有效成分金丝桃素的含量 ,建立了 HPLC测定金丝桃素的新方法 ,该法有良好的重现性及精密度。     

Toxic Effects of Pb on Anatomy and Hypericin Content in Hypericum perforatum L.

Lead (Pb) is one of the most common heavy metal contaminants in the environment. The present study was therefore undertaken to determine the effects of Pb on structural characteristics and hypericin production in Hypericum perforatum. Mature plants were treated with contaminated soil in seven treatments (75, 150, 300, 600, 800, 1000, and 1500 mg/kg Pb in soil) with three repeats per treatment every 14 days. Maximum observed Pb content in shoot parts was observed in the treatments with 600 and 1500 mg/kg Pb. The Pb concentration in roots was higher than in shoot parts, enhanced with increasing Pb concentration in the soil. In this study, Pb treatment significantly influenced the morphology, anatomy, and hypericin content in the plant. Anatomical characteristics of leaf, stem, and root affected by Pb contamination, as well as scanning electron microscopy (SEM) studies, revealed structural changes in stomata and epicuticular waxes. Under Pb toxicity, anatomical symptoms occurred in leaves, including increase in sizes of epidermal cells, mesophyll tissue, and diameter of stems and roots, as well as amplified vascular bundles and pith area. This, therefore, indicated that metal contamination can change the chemical composition of this plant. Maximum hypericin content was observed in the treatment containing 600 mg/kg Pb in soil, which then decreased.     

贯叶连翘中金丝桃素的合成与积累研究进展

金丝桃素是贯叶连翘的主要药理活性成分。本文概述了金丝桃素的化学与生物合成途径,介绍了金丝桃素在贯叶连翘个体发育过程中的积累以及利用贯叶连翘的细胞和组织培养技术生产与积累金丝桃素的研究进展。最后,指出分子生物学和电子显微镜技术的发展为深入研究金丝桃素的产生和积累提供了有利工具。     

Anti-Influenza A Virus Effect of Hypericum perforatum L. Extract

To study the antiviral effect of Hypericum perforatum L. extract (HPE) on influenza A virus (IAV) (H1N1) in vitro and in vivo. Cytopathic effect (CPE) and neutral red (NR) dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney (MDCK) cells which were infected with IAV in vitro. HPE was effective against influenza A virus (IAV) in vitro, with a 50% effective concentration (EC50) of 40 μg/mL. The mean 50% cytotoxic concentration (CC50) in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC50 values of 5.0 μg/mL; the mean CC50 for ribavirin was 520 μg/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin's minimum effective dose was 40 mg/kg/day with the LD50 determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD50 dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza.     

NO和茉莉酸甲酯对黄芩悬浮细胞生长及黄芩苷合成的影响

以硝普钠(sodium nitroprusside,SNP)为一氧化氮(nitric oxide,NO)的供体,向黄芩(Scutellaria baicalensis)悬浮培养细胞系中添加SNP和茉莉酸甲酯(methyl jasmonate,MJ),考察这两种诱导子在不同的添加时间、添加浓度及混合配比使用对黄芩悬浮细胞系生长和黄芩苷含量的影响。研究结果表明：低浓度的外源NO有利于细胞的生长,但对黄芩苷积累无作用,而MJ有利于黄芩苷的合成,但抑制细胞生长,且两者的适用浓度范围和添加时间存在差异。在细胞培养初期(0天)添加0.05 mmol·L~(-1)SNP,而在细胞生长对数中期(8天)添加10μmol·L~(-1)的MJ,细胞鲜重可达到对照的1.2倍,黄芩苷总量达到对照的2.96倍。     

激素对贯叶连翘器官分化的影响

贯叶连翘 (ＨｙｐｅｒｉｃｕｍｐｅｒｆｏｒａｔｕｍＬ .)为多年生草本 ,中国民间主要用于止血、抗炎、妇科病等[1] ,欧洲民间用于治疗创伤也有相当长的历史。近年来 ,欧、美等国家和地区将其应用于抑郁症的治疗 ,取得了很好的疗效。 80年代后期 ,由于发现该植物体内含有显著抗     

贯叶连翘的水培及其代谢产物检测

水培可诱导贯叶连翘组培苗生根能力强,根活力也增加;生根苗在1/6MS培养液中培养6周后的金丝桃素(HP)、假金丝桃素(PHP)和贯叶金丝桃素(HF)含量分别比基质[腐质土 蛭石(1:1)]中培养的提高10.13%、16.00%和61.36%。     

Signal interaction between nitric oxide and hydrogen peroxide in heat shock-induced hypericin production of Hypericum perforatum suspension cells

Heat shock(HS, 40℃, 10 min) induces hypericin production, nitric oxide(NO) generation, and hydrogen peroxide(H2O2) accumulation of Hypericum perforatum suspension cells.Catalase(CAT) and NO spe-cific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(cPTIO) suppress not only the HS-induced H2O2 generation and NO burst, but also the HS-triggered hypericin produc-tion.Hypericin contents of the cells treated with both NO and H2O2 are significantly higher than those of the cells treated with NO alone, although H2O2 per se has no effects on hypericin production of the cells, which suggests the synergistic action between H2O2 and NO on hypericin production.NO treatment enhances H2O2 levels of H.perforatum cells, while external application of H2O2 induces NO generation of cells.Thus, the results reveal a mutually amplifying action between H2O2 and NO in H.perforatum cells.CAT treatment inhibits both HS-induced H2O2 accumulation and NO generation, while cPTIO can also suppress H2O2 levels of the heat shocked cells.The results imply that H2O2 and NO may enhance each other's levels by their mutually amplifying action in the heat shocked cells.Membrane NAD(P)H oxidase inhibitor diphenylene iodonium(DPI) and nitric oxide synthase(NOS) inhibitor S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea(PBITU) not only inhibit the mutually amplifying action between H2O2 and NO but also abolish the synergistic effects of H2O2 and NO on hypericin production, showing that the synergism of H2O2 and NO on secondary metabolite biosynthesis might be dependent on their mutual amplification.Taken together, data of the present work demonstrate that both H2O2 and NO are essential for HS-induced hypericin production of H.perforatum suspension cells.Furthermore, the results reveal a special interaction between the two signal molecules in mediating HS-triggered secondary metabolite biosynthesis of the cells.     

Factors affecting growth of cell suspension cultures of hypericum perforatum L. (St. John's wort) and production of hypericin

Summary Use of Hypericum perforatum L., commonly known as St. John's wort, has increased recently due to the pharmaceutical potential of hypericin, found in its leaves. Hypericin has been reported to effect a natural treatment for mild and moderate depression by increasing the concentration of neurotransmitters in the central nervous system. We have developed a novel cell culture system for in vitro growth and production of this species, suggesting a possible technology for large-scale production of hypericin. Leaf explants grown in Murashige and Skoog salts supplemented with 2,4-dichlorophenoxyacetic acid (0.90 μM) and kinetin (0.11 μM) gave maximum percentage callus formation compared to other medium treatments evaluated. Hypericin localization in cell phase and leaves was found to vary, with cell phase accumulating hypericin in special organelles and leaves accumulating it in vacuoles. Light and dark conditions, with cell aggregate size, played important roles in growth and hypericin production in cell suspension cultures.     

HPLC法测定新疆贯叶连翘中绿原酸和芦丁的含量

采用液相色谱法测定贯叶连翘中绿原酸和芦丁含量。用Hyperil ODS色谱柱(12.5 cm×6.0 mm i.d.,5μm)进行分离。流动相为甲醇-1%冰醋酸梯度洗脱,流速为0.6 mL/min。检测波长为343 nm。绿原酸线性范围为0.0076~0.3040μg,相关系数R=0.9997。芦丁线性范围为0.312~3.120μg,相关系数R=0.9999。样品的平均回收率分别为101.2%,99.8%。此方法准确、快速,适用于绿原酸和芦丁的定量分析。