贯叶连翘愈伤组织中分泌细胞群的发生与金丝桃素类物质的积累

贯叶连翘(Hypericum perforatum L．)是一种传统草药,在欧洲被广泛用于治疗抑郁症。其重要的活性成分,金丝桃素类物质储存在茎、叶和花瓣的分泌细胞团中。本文应用组织化学及电子显微镜技术,研究体外培养的贯叶连翘叶肉细胞脱分化产生愈伤组织以及细胞发育过程中金丝桃素类物质的积累、运输的情况,进一步探讨细胞的生长发育与次生代谢产物的关系。发现金丝桃素类物质产生于愈伤组织培养后期,在愈伤组织表面所形成的分泌细胞群中,最初在细胞质中形成,之后运输至液泡中积累,内质网参与了金丝桃素类物质的合成过程。这些结果为利用组织培养技术提高金丝桃素类物质含量提供了理论基础和依据。     

高压静电场对贯叶连翘种子休眠破除及药用成分的影响

将高压静电场(HVEF)技术应用于贯叶连翘种子休眠破除及组培苗培养,结果显示,贯叶连翘种子在静电场(100 kv/m)中处理1 h,其发芽势(SPO)和发芽率(SPT)是对照组(无静电处理)的5.69倍和2.45倍;贯叶连翘组培苗在静电场(135 kv/m～150 kv/m)中处理1 h,其药用成分总金丝桃素含量和贯叶金丝桃素含量是对照组的1.35～1.53倍;静电场(150 kv/m)处理后,POD活性是其对照的1.30倍.研究表明,一定强度的静电场作用,能有效提高贯叶连翘种子发芽率,促进总金丝桃素和贯叶金丝桃素代谢含量.     

贯叶连翘野生转家化前后生物学性状特征观察

野生贯叶连翘 (HypericumperforatumL .)在江苏经过了 3a的引种栽培 ,其生物学特性检测结果表明 ,栽培贯叶连翘的生物量、开花的一致性和生长的适应性等明显优于野生贯叶连翘 ;良好的繁殖方式使贯叶连翘的收获期提前了 1a ;成熟后 3个月采收的种子萌发率较高 ,不同浓度赤霉素处理能明显提高其种子的萌发率 ;贯叶连翘的黑色腺体即分泌细胞球数量与金丝桃素间也基本存在着正相关关系 ,贵州贵阳居群和陕西丹凤居群黑色腺体明显多于其他居群 ,其金丝桃素含量在所有居群中为最高 ,而甘肃武都居群黑色腺体最少 ,其金丝桃素含量在 6个居群中最低。 6个野生居群栽培后生物量相近 ,在 4个不同土壤pH的基地大田中均能良好生长 ,说明江苏地区环境条件完全适合贯叶连翘引种栽培。     

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%。     

贯叶连翘醇提条件的多指标优化

本研究采用超声波提取法,对贯叶连翘中总黄酮、金丝桃素类和贯叶金丝桃素等主要有效成分在醇提过程中的提取条件(溶媒种类、浓度、提取时间等)进行了考察。结果表明,以总黄酮和金丝桃素类化合物为指标,用65％～80％的乙醇水溶液超声提取30min,提取效率较高;以贯叶金丝桃素为指标,则以用75％～90％的甲醇提取30min效果较好。     

从贯叶连翘叶提取金丝桃素的工艺探讨

对从贯叶连翘中提取金丝桃素的工艺条件进行了优化研究,结果表明,金丝桃素提取较佳工艺为：45℃的水浸泡2h后,加入0.5%NaOH,用75％甲醇在0.05Mpa下78℃提取5次,每次1.5h,溶媒量为5倍,浓缩温度与干燥温度为78℃。     

贯叶连翘抗抑郁研究新进展

贯叶连翘,又名圣约翰草,是传统中药材之一,圣约翰草在德国用于抗抑郁症已有几百年的历史。贯叶连翘提取物对轻度和中度抑郁症患者和动物模型都有很好疗效,最新药理学研究表明贯叶连翘提取物中抗抑郁的主要成分是贯叶金丝桃素。贯叶金丝桃素是神经递质5-羟色胺(5-HT)、多巴胺(DA)、去甲肾上腺素(NE)的非竞争性重吸收抑制剂,贯叶金丝桃素还可以抑制突触体对γ氨基丁酸(GABA)和L-谷氨酸(L-glu)的重吸收,其作用机理至今还不甚明了,研究表明它的作用很可能是通过提高突触体细胞内钠离子浓度或通过降低突触体内突触小泡的跨膜pH梯度实现的。     

贯叶连翘的分泌结构及其与金丝桃素积累的关系

贯叶连翘（ＨｙｐｅｒｉｃｕｍｐｅｒｆｏｒａｔｕｍＬ．）地上器官分布着分泌细胞球（黑色腺点）、分泌囊（半透明腺点）和分泌道（半透明腺条）３类内部分泌结构。分泌细胞球在茎、叶和花器官中均有分布,由２层鞘细胞包围多个分泌细胞构成实心的分泌细胞团。分泌囊主要分布于叶片中,分泌道则分布于花器官中,它们都是由１～２层切向扁平细胞围绕圆形或长形腔道构成,腔道的贮存物为精油。利用组织化学方法,结合荧光显微镜观察,证实金丝桃素类物质是由分泌细胞球（黑色腺点）所合成和积累的。通过用戊二醛和锇酸固定样品的显微和超微结构观察,发现金丝桃素类物质积累在成熟腺体分泌细胞的中央大液泡中,细胞周围浓厚的细胞质中分布着大量小液泡和高尔基体、内质网等细胞器。在此基础上对金丝桃素类物质的积累过程进行了初步探讨     

金丝桃属9种植物种子形态及其系统学讨论

对金丝桃属5组9种植物的种子进行了宏观及微观形态学研究,以探讨岐山金丝桃的系统位置。结果显示,种子形态特征在本实验观察的5个组之间有明显差异。其中金丝桃组种子外形较为细长,种皮纹饰为狭长而规则的矩形网纹;黄海棠组种子稍粗,种皮纹饰为长宽近等的多边形(稀矩形)网纹;元宝草组的元宝草、贯叶连翘组的贯叶连翘以及挺茎遍地金组的云南小连翘种子均较小,元宝草种皮纹饰为负网纹,贯叶连翘种皮纹饰为相邻网纹间有间隙的近圆形网纹,云南小连翘种皮纹饰为复网纹。研究显示,岐山金丝桃的种子形态与黄海棠组最为接近,支持岐山金丝桃归于黄海棠组。     

从贯叶连翘中提取金丝桃素的工艺探讨

对从贯叶连翘中提取金丝桃素的工艺条件进行了优化研究 ,结果表明 ,金丝桃素提取较佳工艺为 :45℃的水浸泡 2 h后 ,加入 0 .5 % Na OH,用 75 %甲醇在 0 .0 5 Mpa下 78℃提取5次 ,每次 1 .5 h,溶媒量为 5倍 ,浓缩温度与干燥温度为 78℃     

NO对金丝桃悬浮细胞生长及金丝桃素生物合成的促进作用研究

一氧化氮 (NO)是近年来发现的一种新型植物信号分子。以硝普钠 (Sodiumnitroprusside ,SNP)为一氧化氮 (NO)的供体 ,研究外源NO对金丝桃悬浮细胞生长及金丝桃素生物合成的影响。试验结果表明 ,金丝桃悬浮细胞在含 0 5和 15 0mmol LSNP的培养基中培养 2 0d后 ,细胞的干重分别为对照组的 140%和50% ;细胞中金丝桃素的含量分别为对照组的 98%和210%。试验结果表明 ,低浓度SNP处理有利于金丝桃悬浮细胞生长 ,而高浓度SNP可以促进金丝桃素的合成。在细胞培养初期 (0d)加入 0.5mmol LSNP并在指数生长后期 (14d)加入15.0mmol LSNP的金丝桃悬浮细胞在培养 2.5d后 ,细胞的干重和金丝桃素的含量分别为对照组的1.4和1.8倍 ,金丝桃素的产量达15.2mg/L ,比对照高3.2倍。SNP对金丝桃悬浮细胞生长及金丝桃素含量的影响可以被NO专一性淬灭剂CPITO(2-4-carboxyphenyl-4 ,4 ,5 ,5-tetramethylimidazoline-1-oxyl-3-oxide)所抑制,说明SNP是通过其分解产物NO影响细胞生长和金丝桃素的合成。试验结果同时表明,在15.0mmol/L的SNP处理下,金丝桃悬浮细胞中的苯丙氨酸解氨酶(PAL)的活性显著升高,推测NO可能通过触发金丝桃悬浮细胞的防卫反应,激活了细胞中金丝桃素的生物合成途径。     

Jasmonic acid-induced hypericin production in cell suspension cultures of Hypericum perforatum L. (St. John's wort)

Hypericum perforatum L. (St. John's wort) is an herbal remedy widely used in the treatment of mild to moderate depression. Hypericin, a photosensitive napthodianthrone, is believed to be the compound responsible for reversing the depression symptoms. In this study, novel in vitro cell culture systems of H. perforatum were used to monitor the effect of elicitation on cell growth and production of hypericin. A dramatic increase in cell growth and hypericin production was observed after exposure to jasmonic acid (JA). However, other elicitors such as salicylic acid (SA) and fungal cell wall elicitors failed to show any stimulatory effect on either cell growth or hypericin production. Cell cultures treated with JA and incubated in the dark showed increased growth and hypericin production as compared to the cultures grown under light conditions. Jasmonate induction in dark conditions played an important role in growth and hypericin production in cell suspension cultures, to our knowledge an undocumented observation.     

Nitric oxide mediates the fungal elicitor-induced hypericin production of Hypericum perforatum cell suspension cultures through a jasmonic-acid-dependent signal pathway

Fungal elicitor prepared from the cell walls of Aspergillum niger induces multiple responses of Hypericum perforatum cells, including nitric oxide (NO) generation, jasmonic acid (JA) biosynthesis, and hypericin production. To determine the role of NO and JA in elicitor-induced hypericin production, we study the effects of NO scavenger 2- to 4-carboxyphenyl-4,4, 5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPITO), nitric oxide synthase inhibitor S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea, and inhibitors of the octadecanoid pathway on elicitor-induced NO generation, JA biosynthesis, and hypericin production. Pretreatment of the cells with cPITO and JA biosynthesis inhibitors suppresses not only the elicitor-induced NO generation and JA accumulation but also the elicitor-induced hypericin production, which suggests that both NO and JA are involved in elicitor-induced hypericin biosynthesis. S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea and cPITO inhibit both elicitor-induced NO generation and JA biosynthesis, while JA biosynthesis inhibitors do not affect the elicitor-induced NO generation, indicating that JA acts downstream of NO generation and that its biosynthesis is regulated by NO. External application of NO via its donor sodium nitroprusside induces hypericin production in the absence of fungal elicitor. Sodium-nitroprusside-induced hypericin production is blocked by JA biosynthesis inhibitors, showing that JA biosynthesis is essential for NO-induced hypericin production. The results demonstrate a causal relationship between elicitor-induced NO generation, JA biosynthesis, and hypericin production in H. perforatum cells and indicate a sequence of signaling events from NO to hypericin production, within which NO mediates the elicitor-induced hypericin biosynthesis at least partially via a JA-dependent signaling pathway.     

Stimulation of the production of hypericins by mannan in Hypericum perforatum shoot cultures

Shoot organ cultures were established from callus derived from anthers of Hypericum perforatum flowers and the effect of elicitors on hypericin and pseudohypericin production in shoot organ cultures was investigated. Mannan stimulated pseudohypericin production up to four fold (0.82 mg/g dry wt) and hypericin production up to two fold (0.04 mg/g dry wt.) beta-1,3-glucan and pectin slightly stimulated pseudohypericin production (ca. two fold), but had no effect on hypericin production. On the other hand, yeast extract showed no stimulatory effect, on either hypericin or pseudohypericin production.     

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.     

用贯叶金丝桃残渣栽培香菇的研究

用提取金丝桃素后的贯叶金丝桃残渣和锯木屑作培养基分别栽培香菇 ,比较了由两种培养基栽培香菇的生物学效率、香菇的可溶性蛋白含量和超氧化物歧化酶 (SOD)活力。结果表明 :用贯叶金丝桃残渣栽培的香菇有较高的生物学效率、其香菇可溶性蛋白含量和SOD活力亦高 ,说明贯叶金丝桃残渣适合栽培香菇 ,为栽培香菇开发了一种新的生物资源。     

Identification and quantification of hypericin and pseudohypericin in different Hypericum perforatum L. in vitro cultures.

Investigations have been made to develop an efficient protocol for micropropagation allowing to improve hypericin and pseudohypericin productions in Hypericum perforatum L. in vitro cultures. The role of growth regulator treatments has been particularly studied. Three in vitro culture lines with different morphological characteristics were obtained during H. perforatum micropropagation and referred to shoots, calli and plantlets according to their appearance. Multiplication and callogenesis from apical segments from sterile germinated seedlings were obtained on solid MS/B5 culture medium in the presence of N6-benzyladenine (BA) (0.1-5.0 mg/l BA). Regenerative potential of shoots was assessed on medium supplemented with auxins (0.05-1.0 mg/l), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). The main goal of the research was to summarize the influence of plant growth regulators on hypericin and pseudohypericin productions in in vitro cultures of Hypericum. A rapid method for naphtodianthrone quantification was developed. The use of a reversed-phase high performance liquid chromatography (HPLC) method with fluorescence detection was used. Identification of the compounds was confirmed by electrospray ionization-mass spectrometry (ESI-MS) with electrospray in negative ion mode [M-H] . Calli, shoots and plantlets of H. perforatum produced hypericin and pseudohypericin. The concentration range of BA from 0.1 to 2.0 mg/l improved the production of hypericin (25-50 microg/g dry mass (DM)) and pseudohypericin (170-350 microg/g DM) in shoots. In callus cultures, BA (4.0-5.0 mg/l) did not changed hypericin contents (15-20 microg/g DM) but influenced pseudohypericin productions (120-180 microg/g DM). In the presence of auxins (IAA and IBA), Hypericum plantlets produced hypericin (30-100 microg/g DM) and pseudohypericin (120-400 microg/g DM). The presence of IAA did not influence naphtodianthrone productions in plantlets, but IBA decreased hypericin and pseudohypericin amounts in plantlets. The specific accumulation of the naphtodianthrones in in vitro cultures was influenced by phytohormonal supplementation of the medium. Results indicated that the production of hypericin and pseudohypericin could be increased by carefully adapted in vitro cultures. Hypericum in vitro cultures represent promising systems for hypericin and pseudohypericin productions.