滇紫草愈伤组织培养的初步研究

滇紫草(Onosma paniculatum)属紫草科滇紫草属植物,其根部皮层富含的紫草素(α—萘醌系列衍生物),可用作治疗外伤与痔疮的药物或高级染料以及化妆品。滇紫草为云南特有,由于人工栽培困难,加之不断采挖,致使这一资源受到严重的破坏,产品供不应求。日本Tabata等对紫草(Lithospermum erythrorhizon)的组织培养进行了一系列的研究,但对滇紫草组织培养以生产紫草素的研究至今未见报道。基于这一现状,我们从1988年开始着手进行滇紫草组织培养的研究,以探讨应用组织培养技术生产紫草素的可能性。     

培养条件对新疆紫草毛状根生长及紫草素含量的影响

以发根农杆菌诱导的新疆紫草毛状根为试验材料,采用二阶段液体培养法,首次建立了新疆紫草毛状根培养技术体系。结果显示:采用SH无铵培养基、pH 5.8时有利于毛状根的生长。培养12d时毛状根的增殖倍数达最高,平均10.26倍;毛状根生产的继代周期为25～30d;4种树脂吸附的紫草素及其衍生物含量均较对照(不添加树脂)高,以NKA-9所吸附的紫草素及其衍生物含量最高,为2.38%,较对照提高0.97倍。培养10d时添加NKA-9树脂,紫草素及其衍生物含量平均为3.64%,是对照的3.08倍。研究表明,生长阶段采用液体培养可以使新疆紫草毛状根快速增殖,生产阶段添加大孔吸附树脂能够提高紫草素及其衍生物含量。     

硬紫草细胞培养色素形成的动力学研究

紫草为我国传统中药,最早收录于《神农本草经》,根部入药。其有效成分为紫红色的萘醌类衍生物的复合物(紫草色素)。它具有抗菌、消炎、抗肿瘤等作用,是具有药用价值的天然色素,在医药、印染、化妆品及食品工业上有广泛的应用前景。借助细胞培养技术生产紫草色素,是开发紫草资源的新途径。1983年日本已将硬紫草细胞培养工业化生产,1984年便有紫草色素产品投放市场。本文测定了硬紫草细胞培养色素形成过程中细胞生长、产物(色素)形     

紫草素及其衍生物的调控策略

紫草素及其衍生物在医药、食品、化妆品和印染等领域有着巨大的市场潜力,如何提高它们的产量已成为该领域研究的热点。综述了调控紫草素及其衍生物的合成和积累方法,主要包括高产细胞系的筛选、生产培养基的改良、外加物和外加条件的影响、基因工程调控、生物反应器的影响及分析和提取纯化技术等,并对有关紫草素及其衍生物今后的发展方向进行了展望。     

中药植物紫草天然产物的生物合成及其功能研究进展

紫草为我国传统的重要药用植物资源,其根部代谢产生的紫红色萘醌类天然产物—紫草素及其衍生物,临床上常被用于治疗疮疡和皮肤炎症。数十年来,紫草因具高效的多重生物活性、药理作用、良好的临床疗效、较高的利用价值,引起了国内外研究者的重视与关注,正由于此种原因,其野生植物种质资源常遭到大量采挖,生长环境受到严重威胁。随着植物天然产物的生物合成、分子代谢及其生物技术的发展,药用植物天然产物生物活性功能与药理作用研究手段的不断创新,紫草的生物合成途径和相关调控基因的研究取得了显著的进展,紫草素药理作用及其机制得到深入阐明或解析,极大地推进了紫草素的基础性研究及其临床应用开发的进程。本文从紫草分类、紫草素的结构与组成及其生物合成途径、调控紫草素生物合成代谢的功能相关基因以及紫草素生物活性与药理功能等方面综述了相关研究进展,并对未来可能的发展趋势进行了展望,以期为促进我国重要中药材源的药用天然产物的深度挖掘与开发提供有益参考,推动我国传统中药学的现代化发展。     

紫草素在消化系统肿瘤中的抗癌机制研究进展

消化系统肿瘤是威胁我国居民生命健康的重要杀手,其发病率和死亡率均占全部肿瘤的50%左右,研发高效安全的抗肿瘤药物是治疗消化系统肿瘤的基础。植物提取物是抗肿瘤药物的重要来源,紫草素(Shikonin)是一种存在于紫草科植物根茎中的药物成分,它对消化系统肿瘤细胞具有显著的杀伤效果。本文通过检索最近10年紫草素在消化系统肿瘤中发挥抗癌作用的相关文献,对紫草素及其衍生物在消化系统肿瘤中的抗癌机制进行系统归纳整理,并分析了今后紫草素应用于临床治疗消化系统肿瘤的研究方向,为进一步探索紫草素在消化系统肿瘤中的抗癌机制研究和新药研发提供理论依据。     

HPLC测定不同产地硬萼软紫草根中4种萘醌类成分的含量

采用HPLC测定10个产地硬萼软紫草根中4种萘醌类成分含量,比较其差异。以乙酰紫草素、去氧紫草素、异丁酰紫草素、(2-甲基正丁酰基)紫草素为对照品,采用Agilent-C_(18)(250 mm×4.6 mm,5μm),以乙腈-0.05%甲酸水溶液(70∶30)为流动相,1 m L/min,检测波长275 nm,柱温30℃。4种成分分离良好,线性、重复性以及回收率均符合要求。不同产地的样品中4种化学成分含量存在差异,可能与植物的生境及采收时间有关,活性成分乙酰紫草素、(2-甲基正丁酰基)紫草素在各样品中含量较高,推断其可作为天然抗肿瘤成分及其衍生物的备选资源,为其资源开发利用提供参考依据。     

新疆紫草细胞大量培养的研究

新疆紫草(Arnebia euchroma)为多年生草本植物,其根中含有多种萘醌类化合物(即紫草宁及其衍生物),为我国传统的中草药,监床用于抗菌、消炎,如治疗皮肤烧伤、烫伤及溃疡久治不愈、妇科炎症等。此外还具有抗促性腺激素及抗肿瘤活性。除药用外,紫草宁衍生物还是理想的天然色素和染料,可用于食品、化妆品的添加剂。     

紫草药用有新方法

紫草药用有新方法紫草为我国传统中草药,南北各地都有,种类繁多,唯有新疆紫草质量最好．中外科技界、医药界研究结果表明,紫草根中富含多种萘醇化合物,即紫草素及其衍生物,临床应用有抗菌抗炎抗病毒抗癌活性;食品应用是理想的天然红色素;日用化工应用在染料、唇膏...     

密花滇紫草萘醌成分研究及紫草素含量测定

从密花滇紫草(Onosma confertum W.W.Smith)根的石油醚提取物中分离出四种化合物,根据其理化常数、光谱数据(MS、IR、~4H-NMR、CD)及 HPLC 等鉴定为:去氧紫草素(Ⅰ)、β,β-二甲基丙烯酰紫草素(Ⅱ)、乙酰紫草素(Ⅲ)和紫草素(Ⅳ)。并对紫草素衍生物碱水解所得紫草素总含量进行了测定。     

Genetic engineering on shikonin biosynthesis: expression of the bacterial ubiA gene in Lithospermum erythrorhizon

The naphthoquinone pigment shikonin from Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) was the first plant secondary metabolite produced in industrial scale from plant cell cultures. We have now manipulated the biosynthetic pathway leading to shikonin in L. erythrorhizon by introduction of the bacterial gene ubiA. This gene of Escherichia coli encodes 4-hydroxybenzoate-3-polyprenyltransferase, a membrane-bound enzyme that catalyzes a key step in ubiquinone biosynthesis. Using geranyl diphosphate (GPP) as substrate, it is able to catalyze the formation of 3-geranyl-4-hydroxybenzoate (GBA), a principal step of shikonin biosynthesis. The prokaryotic ubiA gene was fused to two signal sequences for targeting of the resulting peptide to the endoplasmic reticulum (ER). Constructs with different constitutive promoters were introduced into L. erythrorhizon using Agrobacterium rhizogenes-mediated transformation. In the resulting hairy root lines, high UbiA enzyme activities could be observed, reaching 133 pkat mg(-1). Expression of ubiA resulted in an accumulation of GBA in an amount exceeding that of the control culture by a factor of 50. However, the ubiA-transformed lines showed only a marginal (average 22%) increase of shikonin production in comparison to the control lines, and there was no significant correlation of UbiA enzyme activity and shikonin accumulation. This suggests that overexpression of ubiA alone is not sufficient to increase shikonin formation, and that further enzymes are involved in the regulation of this pathway.     

Molecular cloning and characterization of a cDNA encoding a novel apoplastic protein preferentially expressed in a shikonin-producing callus strain of Lithospermum erythrorhizon

A cDNA (LEPS-2) encoding a novel cell wall protein was cloned from shikonin-producing callus tissues of Lithospermum erythrorhizon by differential display between a shikonin-producing culture strain and a non-producing strain. The LEPS-2 cDNA encoded a polypeptide of 184 amino acids. The deduced amino acid sequence exhibited no significant homology with known proteins. Expression of LEPS-2 gene as well as accumulation of LEPS-2 protein was highly correlated with shikonin production in L. erythrorhizon cells in culture. In the intact plant, expression of LEPS-2 was detected only in the roots where shikonin pigments accumulated. Cell fractionation experiments and immunocytochemical analysis showed that the protein was localized in the apoplast fraction of the cell walls. The shikonin pigments were also stored on the cell walls as oil droplets. These results indicate that expression of the LEPS-2 is closely linked with shikonin biosynthesis and the LEPS-2 protein may be involved in the intra-cell wall trapping of shikonin pigments.     

Ethylene induced shikonin biosynthesis in shoot culture of Lithospermum erythrorhizon.

Lithospermum erythrorhizon shoots, cultured on phytohormone-free Murashige and Skoog solid medium, produced shikonin derivatives, whereas shoots cultured in well-ventilated petri dishes, produced small amount. Analysis by gas chromatography revealed the presence of ethylene in non-ventilated petri dishes where the shoots, producing shikonin derivatives, were cultured. Therefore, the possible involvement of ethylene in shikonin biosynthesis of shoot cultures was investigated. Treatment of ethylene or the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, resulted in increasing shikonin derivatives contents in cultured shoots. Silver ion, an ethylene-response inhibitor, or aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, decreased production of shikonin derivatives in cultured shoots. Our results indicate that ethylene is one of the regulatory elements of shikonin biosynthesis in L. erythrorhizon shoot culture.     

A novel dark-inducible protein, LeDI-2, and its involvement in root-specific secondary metabolism in Lithospermum erythrorhizon

Lithospermum erythrorhizon produces red naphthoquinone pigments that are shikonin derivatives. They are accumulated exclusively in the roots of this plant. The biosynthesis of shikonin is strongly inhibited by light, even though other environmental conditions are optimized. Thus, L. erythrorhizon dark-inducible genes (LeDIs) were isolated to investigate the regulatory mechanism of shikonin biosynthesis. LeDI-2, showing the strict dark-specific expression, was further characterized by use of cell suspension cultures and hairy root cultures as model systems. Its mRNA accumulation showed a similar pattern with that of shikonin. In the intact plants LeDI-2 expression was observed solely in the root, and the longitudinal distribution of its mRNA was also in accordance to that of shikonin. LeDI-2 encoded a very hydrophobic polypeptide of 114 amino acids that shared significant similarities with some root-specific polypeptides such as ZRP3 (maize) and RcC3 (rice). Reduction of LeDI-2 expression by its antisense DNA in hairy roots of L. erythrorhizon decreased the shikonin accumulation, whereas other biosynthetic enzymes, e.g. p-hydroxybenzoic acid:geranyltransferase, which catalyzed a critical biosynthetic step, showed similar activity as the wild-type clone. This is the first report of the gene that is involved in production of secondary metabolites without affecting biosynthetic enzyme activities.     

丹参的冠瘿组织培养和丹参酮的产生

用根癌农杆菌感染丹参无菌苗获得冠瘿组织,除菌后的冠瘿组织在无激素的Ms培养基上生长良好。经高压纸电泳检查,冠瘿组织中含有冠痿碱,证实根癌农杆菌的Ti质粒转化成功。冠瘿组织的生长和丹参酮的积累与基本培养基有关,B5和Ms培养基有利于生长．月增殖倍数分别达到102倍和90倍,而67-V和WP培养基则有利于丹参酮的合成,在培养过程中丹参酮能分泌到培养液中。研究表明用冠瘿组织作为培养系统,生产药用植物有效成分具有良好的开发前景。     

Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene

The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants. The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter. This construct was introduced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformation.The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experiment with [1,7-13C2]shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca. 20% of the overall 4HB biosynthesis in the transgenic cultures. ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.     

美洲商陆毛状根诱导及其离体培养的影响因素

为了探讨利用美洲商陆毛状根生产其药用成分的可能性,研究了美洲商陆毛状根诱导及其离体培养的影响因素。结果表明,美洲商陆叶片外植体被发根农杆菌ATCC 15834感染约18 d后,从其叶片外植体形态学下端叶脉切口处产生毛状根,其中以预培养1 d,农杆菌感染20 min,共培养4 d时的毛状根诱导率最高,达到70%。PCR扩增和硅胶薄层层析结果显示,发根农杆菌Ri质粒的rol C基因以及冠瘿碱合成酶基因已在美洲商陆毛状根基因组中整合和表达。所获得的美洲商陆毛状根系都能在无外源激素的MS固体培养基上快速自主生长;其中以毛状根根系2的生长速度最快、分生侧根能力最强和根表面的根毛密度最高;毛状根根表面呈紫红色或呈白色。在供试的MS、1/2MS、B5和6,7-V液体培养基中,以无外源激素的MS培养基最适合美洲商陆毛状根根系生长。与无外源激素的MS培养基相比,6,7-V培养基更有利于毛状根中商陆皂苷甲的合成与积累。本文所建立的美洲商陆毛状根诱导及其离体培养的适宜条件为今后利用其毛状根株系的规模培养来生产其药用有效成分商陆皂苷甲奠定了实验和技术基础。     

Physiology of Hormone Autonomous Tissue Lines Derived From Radiation-Induced Tumors of Arabidopsis thaliana

γ-Radiation-induced tumors of Arabidopsis thaliana L. have been produced as a novel approach to isolation of genes that regulate plant development. Tumors excised from irradiated plants are hormone autonomous in culture and have been maintained on hormone-free medium for up to 4 years. Five tumor tissue lines having different morphologies and growth rates were analyzed for auxin, cytokinin, and 1-aminocyclopropane-1-carboxylic acid (ACC) content, ethylene production, and response to exogenous growth regulators. Normal tissues and two crown gall tissue lines were analyzed for comparison. Rosettes and whole seedlings each contained approximately 30 nanograms· (gram fresh weight)⁻¹ free indoleacetic acid (IAA), 150 nanograms· (gram fresh weight)⁻¹ ester-conjugated IAA, and 10 to 20 micrograms· (gram fresh weight)⁻¹ amide-conjugated IAA. The crown gall lines contained similar amounts of free and ester-conjugated IAA but less amide conjugates. Whereas three of the radiation-induced tumor lines had IAA profiles similar to normal tissues, one line had 10- to 100-fold more free IAA and three- to 10-fold less amide-conjugated IAA. The fifth line had normal free IAA levels but more conjugated IAA than control tissues. Whole seedlings contained approximately 2 nanograms· (gram fresh weight)⁻¹ of both zeatin riboside and isopentenyladenosine. The crown gall lines had 100- to 1000-fold higher levels of each cytokinin. In contrast, the three radiation-induced tumor lines analyzed contained cytokinin levels similar to the control tissue. The radiation-induced tumor tissues produced very little ethylene, although each contained relatively high levels of ACC. Normal callus contained similar amounts of ACC but produced several times more ethylene than the radiation-induced tumor lines. Each of the radiation-induced tumor tissues displayed a unique set of responses to exogenously supplied growth regulators. Only one tumor line showed the same response as normal callus to both auxin and cytokinin feeding. In some cases, one or more tumor lines showed increased sensitivity to certain growth substances. In other cases, growth regulator feeding had no significant effect on tumor tissue growth. Morphology of the radiation-induced tumor tissues generally did not correlate with auxin to cytokinin ratio in the expected manner. The results suggest that a different primary genetic event led to the formation of each tumor and that growth and differentiation in the tumor tissue lines are uncoupled from the normal hormonal controls.     

High level expression of chorismate pyruvate-lyase (UbiC) and HMG-CoA reductase in hairy root cultures of Lithospermum erythrorhizon

Shikonin, a red naphthoquinone pigment, is produced by cell cultures of Lithospermum erythrorhizon (Boraginaceae). It is biosynthetically derived from two key precursors, 4-hydroxybenzoate (4HB) and geranyldiphosphate (GPP). The bacterial ubiC gene, encoding chorismate pyruvate-lyase (CPL) which converts chorismate to 4-hydroxybenzoate, was expressed in L. erythrorhizon under the control of the strong (ocs)(3)mas-promoter. This introduced an efficient biosynthetic pathway to 4HB, i.e. a one-step reaction from chorismate, in addition to the endogeneous multi-step phenylpropanoid pathway. Feeding experiments with [1,7-(13)C(2)]shikimic acid showed that in the most active transgenic line, 73% of 4HB was synthesized via the genetically introduced pathway. However, there was no correlation between CPL activity and 4HB glucoside or shikonin accumulation in the transgenic lines. HMG-CoA reductase (HMGR) is involved in the biosynthesis of GPP in L. erythrorhizon. Two forms of HMGR1 of Arabidopsis thaliana were expressed in Lithospermum under control of the (ocs)(3)mas promoter. Only moderate increases in enzyme activity were obtained with the complete enzyme, but high activity was achieved using the soluble cytosolic domain of HMGR1. Shikonin accumulation remained unchanged even upon high expression of soluble HMGR.     

Functional Expression of Agrobacterium tumefaciens T-DNA onc-genes in Asparagus Crown Gall Tissues

We analyzed Asparagus crown gall tissues transformed with A.tumefaciens C58 and C58C1 (pTiB6S3) and selected for hormoneautotrophic growth. No increased IAA levels were observed inthe Asparagus tumor lines notwithstanding the presence of allthree T-DNA onc genes. The endogenous cytokinin levels indicatethat Asparagus crown gall is dependent on enhanced zeatin ribosideequivalent levels for its growth. We conclude that phytohormone autotrophic growth of Asparaguscrown gall tissue seems only to be dependent upon an activegene 4, inducing enhanced cytokinin levels. Moreover, the presenceof an active gene 1 seems to be lethal as was indicated by theabsence of tryptophan-2-mono-oxygenase activity in transformedtissues and the toxicity of exogenously supplied indole-3-acetamide(IAM) or naphthalene-1-acetamide (NAM) as a substitute for anactive gene 1. (Received August 7, 1989; Accepted October 31, 1989)