喜树碱和10-羟基喜树碱受喜树生长发育调控的不同特点

为了揭示喜树碱(camptothecin, CPT)和10-羟基喜树碱(10-hydroxycamptothecin, HCPT)在喜树(Camptotheca acuminata Decaisne)体内代谢的生理调控机制及这两种类似物之间的关系和作用,运用高效液相色谱技术对种子形成、成熟、萌发和幼苗生长过程中喜树碱和10-羟基喜树碱的代谢动态进行了全面的研究.结果表明,喜树碱相对稳定地存在于成熟和幼嫩的组织中;10-羟基喜树碱特异性地积累在乳熟期的种子、种芽的子叶、幼嫩的真叶等幼嫩组织中,随着组织的进一步发育和成熟,其含量快速减少,成熟组织中积累极少,并且这两种生物碱的代谢在时间和数量上都呈现出相互消长的特点,由此推断,这两种生物碱不同程度地受生长发育调控的特点可能与喜树的化学防御策略密切相关,并且,它们之间可以相互转化以充分利用有限的氮素资源.     

喜树幼苗中喜树碱和10-羟基喜树碱对热激的响应特点和意义

植物在长期的生态环境适应过程中,产生了包括生物碱在内的大量次生代谢物。本文以我国特有树种——喜树(Camptotheca acuminata Decaisne)为材料,研究其不同器官中喜树碱(camptothecin,CPT)和10-羟基喜树碱(10-hydroxycamptothecin,HCPT)在不同热激温度和时间情况下的含量变化。CPT和HCPT变化呈现出较好的相互消长关系,并且分别在38℃和40℃达到各自的峰值,比以丙二醛和叶绿素为指标的致死温度低了2-4℃：HCPT在热激过程中的变化较CPT活跃;极易受到攻击和伤害的嫩叶中的生物碱含量变化最大。由此推断,CPT和HCPT遵循“幼嫩和生殖器官优先保护”的原则,从而有效地缓解了高温胁迫,并且HCPT和CPT代表了不同的防御策略。     

喜树幼苗中喜树碱和10-羟基喜树碱对热激的响应特点和意义

植物在长期的生态环境适应过程中,产生了包括生物碱在内的大量次生代谢物.本文以我国特有树种--喜树(Camptotheca acuminata Decaisne)为材料,研究其不同器官中喜树碱(camptothecin,CPT)和10-羟基喜树碱(10-hydroxycamptothecin,HCPT)在不同热激温度和时间情况下的含量变化.CPT和HCPT变化呈现出较好的相互消长关系,并且分别在38℃和40℃达到各自的峰值,比以丙二醛和叶绿素为指标的致死温度低了2~4℃;HCPT在热激过程中的变化较CPT活跃;极易受到攻击和伤害的嫩叶中的生物碱含量变化最大.由此推断,CPT和HCPT遵循"幼嫩和生殖器官优先保护"的原则,从而有效地缓解了高温胁迫,并且HCPT和CPT代表了不同的防御策略.     

抗癌药物喜树碱生产工艺的研究

前言抗癌药物喜树碱是含在我国南方的一种特产值物——珙桐科喜树(Camptothecaacuminata Decne.)中的一种生物碱,其结构为:在喜树的木、根皮和种子中都含有,以种子中台量为最高,主要成伪一为喜树碱 (c amPtothecine),其他还有10一径基喜树碱     

喜树碱的生物合成途径和代谢调控

喜树碱是一种具有抗肿瘤活性的萜类吲哚生物碱,最早是从我国特有植物喜树中分离获得的。本文概述喜树碱的天然分布合成途径和代谢调控的研究进展,并对喜树碱未来的生产和发展研究作了展望。     

不同产地和生长季节喜树叶中喜树碱含量测定

喜树（Camptotheca acuminata）是我国特有抗癌植物。本文通过测定不同产地的喜树幼嫩叶和成熟叶中喜树碱含量表明,均以四川都江堰所产喜树叶中喜树碱含量最高。不同生长月份的喜树嫩叶中喜树碱含量成一条单峰曲线,以7月喜树嫩叶中喜树碱的含量最高。     

不同产地和生长季节喜树叶中喜树碱含量测定

喜树(Camptotheca acuminata)是我国特有抗癌植物。本文通过测定不同产地的喜树幼嫩叶和成熟叶中喜树碱含量表明,均以四川都江堰所产喜树叶中喜树碱含量最高。不同生长月份的喜树嫩叶中喜树碱含量成一条单峰曲线,以7月喜树嫩叶中喜树碱的含量最高。     

喜树果中喜树碱和10-羟基喜树碱的匀浆提取工艺

以喜树果为原料,对匀浆法提取喜树碱和10-羟基喜树碱的工艺进行了研究,确定了最佳的工艺条件：提取溶剂为体积分数55％的乙醇,匀浆时间为8min,料液比为1：15（g／mL）。在此工艺条件下,喜树碱和10-羟基喜树碱的提取率分别为0．801‰和0．546‰。将该法与超声提取、回流提取、常温冷浸提取、水浴振荡提取进行了比较,结果表明,匀浆提取具有得率高、省时间等方面的优势,是一种高效提取喜树碱和10．羟基喜树碱的方法。     

丛枝菌根对喜树幼苗喜树碱含量的影响

喜树（Camptotheca acuminata）是我国特有的多年生亚热带落叶阔叶树种,因其次生代谢产物喜树碱具有良好的抗肿瘤活性而受到人们的广泛关注.通过温室盆栽接种试验,观察了2属6种丛枝菌根真菌对喜树幼苗喜树碱含量的影响.结果表明,接种的6种丛枝菌根真菌与喜树幼苗均形成了共生体系并且发育良好.透光球囊霉（Glomus diaphanum）、幼套球囊霉（G.etunicatum）、蜜色无梗囊霉（Acaulospora mellea）和光壁无梗囊霉（A.laevis）侵染形成丛枝菌根有利于提高喜树幼苗的喜树碱含量,地表球囊霉（G.versiforme）则影响不大,而木薯球囊霉（G.manihot）却降低了喜树幼苗的喜树碱含量.丛枝菌根形成对喜树幼苗喜树碱代谢的影响还表现在喜树碱的器官分配上,菌根幼苗根中的喜树碱比例均高于无菌根幼苗.     

接种时期对丛枝菌根喜树幼苗喜树碱含量的影响

喜树(Camptotheca acuminata)是我国特有的多年生亚热带落叶阔叶树种, 因其次生代谢产物喜树碱具有良好的抗肿瘤活性而受到人们的广泛关注。通过温室盆栽接种试验, 观察了喜树幼苗不同生长时期接种蜜色无梗囊霉(Acaulospora mellea)和根内球囊霉(Glomus intraradices)对喜树幼苗喜树碱积累的影响。结果表明接种两种丛枝菌根真菌均促进了喜树幼苗喜树碱的积累, 表现为喜树碱产量(单株幼苗所含的喜树碱量, 喜树碱含量与幼苗生物量的乘积)的显著提高。进一步分析发现, 接种丛枝菌根真菌导致幼喜树苗喜树碱产量的提高, 早期(幼苗出土20天)接种主要是源于喜树碱含量的提高, 特别是叶片喜树碱含量的提高, 而晚期(幼苗出土60天)接种则主要是源于幼苗生物量的增加。     

Camptothecin and 10-hydroxycamptothecin accumulation in tender leaves of Camptotheca acuminata saplings after treatment with plant growth regulators

The effect of plant growth regulators (PGRs) on the accumulation of the alkaloid camptothecin (CPT) and its analogue 10-hydroxycamptothecin (HCPT) in tender leaves of Camptotheca acuminata saplings was studied. In screening experiments for PGRs, 40?mg/L dose of thiourea, triacontanol, and ascorbic acid (V_C) had no positive effects on the accumulation of the alkaloids. However, treatments with 40?mg/L of chlormequat chloride (CCC), choline chloride, paclobutrazol (PBZ), and daminozide (B₉) induced CPT and HCPT accumulation in both pre-harvest and postharvest stages. On that basis, five levels of PGRs at 0, 20, 40, 60, 80?mg/L were sprayed on tender leaves of C. acuminata saplings at pre-harvest and postharvest stages. Treatment by 40?mg/L CCC dramatically enhanced HCPT production by 308?% in pre-harvest, treatment by 60?mg/L CCC enhanced HCPT production by 100?% in postharvest. Spraying the leaves with 60?mg/L choline chloride resulted in 94?% increase of CPT and spraying with 40?mg/L of the PGRs reached 167?% increase of HCPT in the pre-harvest treatment, respectively; treatments with 60?mg/L choline chloride resulted in 64?% increase of CPT and 525?% increase of HCPT in postharvest, respectively. 52?% increase of CPT and 86?% increase of HCPT in pre-harvest, 22?% increase of CPT and 33?% increase of HCPT in postharvest were obtained by spraying leaves with 60?mg/L PBZ. Treatments with 40?mg/L B₉ had the highest impact on CPT (12?% increase in pre-harvest, 11?% increase in postharvest) and HCPT (167?% increase in pre-harvest, 173?% increase in postharvest) accumulation. The optimal PGR for obtaining the highest levels of CPT and HCPT was treatment with 60?mg/L choline chloride. In most case, the pre-harvest treatment was better than the postharvest one. These preliminary results suggest that the application of PGRs may be a useful and feasible method to increase CPT and HCPT levels in C. acuminata.     

Extraction and composition of three naturally occurring anti-cancer alkaloids in Camptotheca acuminata seed and leaf extracts

Naturally occurring camptothecins (CPT) are important sources of chemotherapeutic agents for clinical treatment of cancer. Extraction of CPT from Camptotheca acuminata trees remains to be a cost-effective way in the supply equation compared with a total synthesis. This study conducted a series of experiments to determine efficient solvent for the maximal extraction of CPT and its two derivatives, hydroxycamptothecin (HCPT) and methoxycamptothecin, from seeds and leaves of C. acuminata. Methanol as an extraction solvent demonstrated in seeds a significantly higher recovery of these three alkaloids than dichloromethane and acetone. Methanol concentrations at 70% in water resulted in maximum extraction of all the three alkaloids regardless of the type of plant materials. However, other strengths of methanol, lower or higher, either decreased the extracting power or showed no improvement in the extraction. Seed extract contained all the three alkaloids whereas leaf extract was absent of HCPT. A stable ratio of the three alkaloids was discovered but it was dependent upon seed or leaf extract of C. acuminata, which with various compositions can be produced. Ecological and medicinal implications of the leaf and seed extract characterized with different chemical compositions are discussed.     

Characterization of DNA Topoisomerase-1 in Spodoptera exigua for Toxicity Evaluation of Camptothecin and Hydoxy-Camptothecin

Camptothecin (CPT), a plant alkaloid originally isolated from the native Chinese tree, Camptotheca acuminate, exerts the toxic effect by targeting eukaryotic DNA topoisomerase 1 (DNA Topo1). Besides as potent anti-cancer agents, CPT and its derivatives are now being explored as potential pesticides for insect control. In this study, we assessed their toxicity to an insect homolog, the Topo1 protein from beet armyworms (Spodoptera exigua Hübner), a worldwide pest of many important crops. The S. exigua Topo1 gene contains an ORF of 2790 base pairs that is predicted to encode a polypeptide of 930 amino acids. The deduced polypeptide exhibits polymorphism at residue sites V420, L530, A653 and T729 (numbered according to human Topo1) among insect species, which are predicted to confer sensitivity to CPT. The DNA relaxation activity of this protein was subsequently examined using a truncated form that contained the residues 337–930 and was expressed in bacteria BL21 cells. The purified protein retained the ability to relax double-stranded DNA and was susceptible to CPT and its derivative hydroxy-camptothecin (HCPT) in a dose-dependent manner. The same inhibitory effect was also found on the native Topo1 extracted from IOZCAS-Spex-II cells, a cell line established from beet armyworms. Additionally, CPT and HCPT treatment reduced the steady accumulation of Topo1 protein despite the increased mRNA expression in response to the treatment. Our studies provide information of the S. exigua Topo1 gene and its amino acid polymorphism in insects and uncover some clues about potential mechanisms of CPT toxicity against insect pests. These results also are useful for development of more effective Topo1-targeted CPT insecticides in the future.     

Hydroxycamptothecin-induced apoptosis in hepatoma SMMC-7721 cells and the role of mitochondrial pathway

The camptothecin (CPT) derivative hydroxycamptothecin (HCPT) containing 10-hydroxy represents one of the most potent topoisomerase I inhibitors described. This anticancer agent, currently undergoing clinical trials on gastric tumours, has been shown more active and less toxic than conventional camptothecins. To shed light on the mechanism of action of HCPT at the cellular level, we examined cell growth, apoptosis, changes of mitochondrial membrane potential, cytochrome c and AIF translocation in cancer cells by exposing these cells to HCPT for indicated time. The effect of HCPT on cell proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay and apoptosis was measured using flow cytometry, fluorescence microscopy and electron microscopy. Changes of mitochondrial membrane potential were monitored by fluorescence microscope. Western blot analysis was used to evaluate the release of mitochondrial cytochrome c and AIF; On the other hand, translocation of cytochrome c and AIF from mitochondria to cytosol during apoptosis were confirmed by confocal microscopy. HCPT could noticeably inhibit the proliferation of SMMC-7721cells and the IC(50) dose was about 0.22muM; SMMC-7721 cells treated with HCPT showed typical characteristics of apoptosis rather than necrotic including phosphatidylserine (PS) exposed from the inner to the outer leaflet of the plasma membrane, abnormal cell morphology, chromatin condensation and nuclear fragmentation; On the other hand, during process of cell apoptosis, mitochondrial transmembrane potential was reduced; Compared with the control group, the mRNA and protein expression of cytochrome c and AIF in treated and untreated SMMC-7721 cells were not significantly changed (not shown). However, when cells were treated with HCPT, the massive translocation of cytochrome c and AIF to the nucleus was evident. Our results indicate that HCPT can inhibit proliferation and induce apoptosis of human hepatoma SMMC-7721 cells. Mitochondrial pathway of apoptosis, especially for cytochrome c and AIF translocation, may play an important role in apoptosis induced by HCPT.     

Camptothecin, over four decades of surprising findings

Camptothecin (CPT) is a modified monoterpene indole alkaloid produced by Camptotheca acuminata (Nyssaceae), Nothapodytes foetida, Pyrenacantha klaineana, Merrilliodendron megacarpum (Icacinaceae), Ophiorrhiza pumila (Rubiaceae), Ervatamia heyneana (Apocynaceae) and Mostuea brunonis (Gelsemiaceae), species belonging to unrelated orders of angiosperms. From the distribution of CPT and other secondary metabolites, it has been postulated that the genes encoding enzymes involved in their biosynthesis evolved early during evolution. These genes were presumably not lost during evolution but might have been "switched off" during a certain period of time and "switched on" again at some later point. The CPT derivatives, irinotecan and topotecan, are used throughout the world for the treatment of various cancers, and over a dozen more CPT analogues are currently at various stages of clinical development. The worldwide market size of irinotecan/topotecan in 2002 was estimated at about $750 million and at $1 billion by 2003. In spite of the rapid growth of the market, CPT is still harvested by extraction from bark and seeds of C. acuminata and N. foetida. All parts of C. acuminata contain some CPT, although the highest level is found in young leaves (approximately 4-5 mg g(-1) dry weight), approximately 50% higher than in seeds and 250% higher than in bark. The development of hairy root cultures of O. pumila and C. acuminata, and the cloning and characterization of genes encoding key enzymes of the pathway leading to CPT formation in plants has opened new possibilities to propose alternative and more sustainable production systems for this important alkaloid.     

Camptothecin and 10-hydroxycamptothecin from<Emphasis Type="Italic"> Camptotheca acuminata</Emphasis> hairy roots

Camptothecin (CPT) is an anticancer and antiviral alkaloid produced by the Chinese tree Camptotheca acuminata (Nyssaceae) and some other species belonging to the families Apocynaceae, Olacaceae, and Rubiaceae. Bark and seeds are currently used as sources for the drug. Several attempts have been made to produce CPT from cell suspensions; however, the low yields obtained limit this approach. Cultures of differentiated cell types may be an alternative source of alkaloid production. Hairy root cultures of C. acuminata were established from tissue transformed with Agrobacterium rhizogenes strains ATCC 15834 and R-1000. Integration of the genes responsible for the hairy-root phenotype (rol genes) into the plant genome was verified by DNA gel blot analysis. The hairy roots produce and secrete CPT as well as the more potent and less toxic natural derivative, 10-hydroxycamptothecin (HCPT), into the medium. Remarkably, the cultures were able to synthesize the alkaloids at levels equal to, and sometimes greater than, the roots in planta, i.e., 1.0 and 0.15 mg/g dry weight for CPT and the HCPT, respectively.Abbreviations CPT Camptothecin - DR Dry weight - FW Fresh weight - HCPT 10-Hydroxycamptothecin - HPLC High-performance liquid chromatography - kb Kilobases - NAA 1-Napththalenacetic acid - TLC Thin-layer chromatographyCommunicated by K.K. Kamo     

Camptothecin accumulation in various organ cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne grown in different culture systems

Levels of camptothecin (CPT) and 10-hydroxycamptothecin (HCPT) were determined in different cultures of Camptotheca acuminata grown either in a Temporary Immersion System (TIS) or on solid medium. CPT was also detected in liquid culture medium. HPLC analysis showed significant differences in CPT contents in all tissues analysed and the highest CPT contents were found in shoots grown on solid medium and in TIS with a mean of 2.2 and 2.5 mg g⁻¹ DW, respectively. The highest content of CPT detected in seedlings was 1.96 mg g⁻¹ DW; while that of somatic embryos at cotyledonary stage and regenerated plants were 0.87 and 1.23 mg g⁻¹ DW, respectively. It was also shown that shoots cultured in TIS secreted substantial amount of CPT into the liquid medium. After 4 weeks in culture a mean of 6, 05 and 12, 6 μg g⁻¹ FW were determined at 4 and 8 immersion cycles daily (IC d⁻¹), respectively. This aspect opens new possibilities regarding the isolation of CTP using TIS culture systems.     

Drought-induced in vivo synthesis of camptothecin in Camptothecaacuminata seedlings

Biosynthesis of secondary metabolites may be affected by environmental stimuli. In the present work, the effect of drought on the levels of an indole alkaloid (camptothecin [CPT]) in Camptotheca acuminata seedlings was investigated. Three seed sources, one from its native habitat in China and two from earlier introductions to the United States, in Texas and South Carolina, were used to compare response patterns. Progressive drought stress significantly reduced biomass production in the 3 seed sources of C. acuminata . Stomatal conductance closely followed the drought cycles, indicating the stress levels experienced by the plants. Leaf CPT concentrations showed a strong increase in the initial drought cycle in all seed sources except Texas, but they deviated in the second drought cycle, in which the South Carolina source continued to increase, whereas the Chinese and Texas seed sources decreased. CPT was inducible by drought, but the sustainability of the drought effect on leaf CPT concentrations was low and varied among seed sources.