Relation between dissolved oxygen concentration and ajmalicine production rate in high-density cultures of Catharanthus roseus

The relation between dissolved oxygen (DO) and the ajmalicine production rate of Catharanthus roseus was investigated in 15-L tank reactors at constant stirrer speed and gas flow rate. Below a DO concentration of 29% of air saturation the ajmalicine production rate was less than 0.06 mumol/g/d. Above a DO of 43% the ajmalicine production rate was constant at 0.21 mumol/g/d. Between a DO of 29% and 43% there was a strong relation between the ajmalicine production rate and the DO concentration. After a period of at least 12 days at DO /=57%. A kinetic equation is proposed for the relation between DO and the specific ajmalicine production rate. (c) 1995 John Wiley & Sons, Inc.     

Scaleup of ajmalicine production by plant cell cultures of Catharanthus roseus

The effect of scaleup on he production of ajmalicine by a Catharanthus roseus cell suspension culture in a selected induction medium were studied. In preliminary experiments it was observed that the culture turned brown and the production was inhibited upon transfer from a shake flask to a stirred bioreactor with forced aeration. Two factors were recognized as the potential origin of the differences between shake flask and bioreactor cultures: gas composition and mechanical shear forces. These factors were studied separately.By recirculating a large part of the exhaust gas, a comparable gas regime was obtained in a bioreactor as occurred in a shake flask cultures. This resulted in the absence of browning and a similar pattern of ajmalicine production as observed in shake flasks. The effect of shear forces could not be demonstrated. However, the experiments showed that the culture may be very sensitive to liquid phase concentrations of gaseous compounds. The effects of k(L)a, aeration rate, CO(2) production rate, and influent gas phase CO(2) concentration on the liquid phase CO(2) concentration are discussed. (c) 1993 John Wiley & Sons, Inc.     

Effects of oxygen and nutrients limitation on ajmalicine production and related enzyme activities in high density cultures of Catharanthus roseus

Oxygen and nutrient limitation was investigated in order to identify the origin of a lower specific ajmalicine production in Catharanthus roseus cultures at high cell densities in an induction medium. The effect of oxygen limitation was explored by comparing two identically aerated and agitated high cell density bioreactor cultures with dissolved oxygen (DO) concentration of 15% and 85% of air saturation, with respect to alkaloid formation and related enzymes activities. Oxygen had an evident effect on ajmalicine production: in the high DO cultures production was more than 5 times higher than in the low DO cultures. The difference in ajmalicine production between high and low DO could not be explained by the enzyme activity profiles. Moreover, the productivity in the high density culture could not restored to the level of a low density culture (at a high DO) by increasing the DO alone. The effect of nutrient limitation was studied with response surface methodology in shake flask cultures. Nutrient limitation could not be demonstrated to be responsible for the productivity loss. Alkaloid and enzyme measurements in the shake flask cultures supported previous findings that the tryptamine pathway may regulate alkaloid production, provided that the terpenoid pathway is sufficiently active. (c) 1994 John Wiley & Sons, Inc.     

Studies on the optimization of growth and indole alkaloid production by hairy root cultures of Catharanthus roseus

Catharanthus roseus hairy root cultures, genetically transformed with Agrobacterium rhizogenes, produce a wide variety of indole alkaloids. The effect of sucrose, phosphate, nitrate, and ammonia concentrations on growth and indole alkaloid production of C. roseus hairy root cultures were studied by using statistical experimental designs and linear regression analysis. Contradictory effects of these nutrients on growth and indole alkaloid production were found. The maximal growth was obtained by having 77. 8 mg NaH(2)PO(4) . H(2)O/L and 1. 311 g KNO(3)/L in the medium, whereas the specific production of alkaloids was highest at the lowest levels of all the nutrients studied. The maximal dry weight was obtained with high values of sucrose and ammonia, but clear optimum concentrations could not be found. When having enough nutrients to support reasonable growth, it appeared difficult to affect the specific alkaloid production rates considerably. The growth (dry wt.) with the optimized nutrient concentrations in the medium was more than 50% better than in the control medium with about the same alkaloid production.     

长春花(Catharanthus roseus)中吲哚类生物碱含量的比较

建立了反相高效液相法测定长春花中吲哚类生物碱文多灵、长春质碱和阿玛碱含量的方法,色谱柱为HiQ sil C18色谱柱（250 mm×4.6 mm,5 μm）;流动相为1%二乙胺水溶液(磷酸调pH=7.2)—甲醇—乙腈/2:1:1(V/V);流速为1 mL·min^-1;检测波长为215 nm;柱温为40℃。并采用此方法对长春花根、茎、叶、花和种子以及不同产地的长春花中的这3种生物碱进行了检测,结果表明文多灵和长春质碱主要存在于在根、茎、叶、花中,阿玛碱主要存在于种子中;并且随着地理位置的北移,长春花中的文多灵和长春质碱的含量逐渐降低,温室中人为控制长春花中的栽培条件能提高二者的含量。     

Nutrient consumption and alkaloid accumulation in a hairy root line of Catharanthus roseus

A hairy root line from Catharanthus roseus was cultured in a 14 l bioreactor. Nitrate and phosphate uptakes were similar to the same root line cultured in a 250 ml Erlenmeyer flask. However, sucrose consumption rate was slower in roots cultured in the bioreactor. These results show that it is feasible to upgrade this hairy root line to bioreactor level and, although carbon utilization has to be improved, ajmalicine and catharanthine were still produced and were retained within the biomass tissue.     

植物生长调节物质对长春花细胞中吲哚生物碱积累的影响

适当浓度的脱落酸(ABA)、乙烯利和乙酰水杨酸(ASA)均对长春花悬浮细胞中吲哚生物碱的积累有较明显的促进作用.2mg·L-1ABA对吲哚生物碱的诱导效果最好,乙烯利的最适剂量为1 g·L-1,1 mg·L-1ASA有利于阿玛碱积累,而2 mg·L-1ASA则有利于长春质碱积累.     

外源钾水平对长春花生长和生物碱积累的影响

钾营养对于植物的生长发育具有重要作用,通过对长春花施以不同水平的外源钾,以探讨在不同浓度的钾素营养条件下,长春花生物量和生物碱的积累特点。结果表明,外源钾素营养水平的增加显著促进长春花的株高、根长、生物量积累,其中以10和15 mmol·L^-1浓度下的作用效果最显著。同时,外源的钾素营养水平提高还显著增加长春花的吲哚类生物碱含量,其中长春质碱的含量随着钾素营养浓度增大而增加;文多灵和长春碱随着钾素营养浓度增大都是先增加后减小,在15 mmol·L^-1浓度下达到最大值,过高的钾素营养不利于两者的合成。     

刈割对栽培长春花(Catharanthus roseus)生活史型转变的影响

为了提高栽培长春花（Catharanthus roseus）的生物碱产量,应用植物生活史型的理论和方法,研究了刈割对栽培长春花生活史型转变及其生物碱代谢的影响。运用主成分分析法（Principal Component Analysis,PCA）对刈割后的长春花后生活史型变化进行定量和定性划分,发现在对照栽培环境下生长的长春花处于DE生境,定性划分结果为SV生活史型,定量划分结果为V0.3638,S0.6174C0.0187,属于SV型。刈割使长春花的生活史型转变为V0．2847S0.6544C0.0469,属于SC型。同时,对两种生活史型的长春花中长春碱及其前体文朵灵和长春质碱的含量进行了检测分析,发现刈割后的SC型长春花不同叶位叶片中的生物碱含量均显著提高（P〈O．05）,可以为提高栽培长春花生物碱含量提供科学指导,也进一步验证了生活史型理论。     

长春花生物碱成分及其药理作用研究进展

本文综述了长春花的研究进展,并着重介绍了长春花生物碱成分、药理作用的研究进展。     

长春花激素完全适应型细胞的生长和阿玛碱合成特性

从长春花激素依赖型细胞系（C20D）筛选出一种激素完全适应型的细胞系（C20hi),考察了两种细胞生长、阿玛碱合成和引吲哚生物碱生物合成相关的酶的活性,结果表明：在生长培养基上二生长无显差异,而C20hi细胞平均阿玛碱含量是C20D的31.9倍,在生产培养基上C20hi细胞生长较C20D快,C20hi平均阿玛碱含量是C20D的18.4倍。通过比较生产和生长培养基中C20hi细胞的色氨酸脱羧酶、异胡豆苷合酶和long牛儿醇-10-脱氢酶活性,发明,通过5年的继代培养,激素完全适应型细胞系C20hi的阿玛碱含量是比较稳定的。     

Enhancement of ajmalicine production in Catharanthus roseuscell cultures with methyl jasmonate is dependent on timing and dosage of elicitation

The optimum growth stage for enhancing ajmalicine production in Catharanthus roseus cultures with methyl jasmonate (MJ) was after 6 d growth. MJ added at 10 or 100 microm on day 6 gave a maximum ajmalicine production of 10.2 mg l(-1), a 300% increase over that of non-elicited cultures.     

Alginate promotes production of various enzymes by Catharanthus roseus cells

When 1.0 g/l of alginate was added to a Catharanthus roseus L. cell culture, many proteins were released from the cells as detected by SDS polyacrylamide gel electrophoresis. In particular, production and/or release of 5′-phosphodiesterase (5′-PDase), catalase and chitinase by C. roseus L. cells were promoted by the addition of alginate. The promotive effects of alginate on 5′-PDase production were observed for various C. roseus cell lines and similar results were obtained when different alginates with various mannuronate/guluronate ratios and viscosities were used. In contrast, agar, agarose, and chitosan did not promote 5′-PDase production. The promotion of 5′-PDase production was not due to cell mutation, the alginate acted rather as a kind of elicitor. During 82 subcultures (577 days) in Murashige and Skoog medium containing 1.0 g/l of alginate, production and release of 5′-PDase by C. roseus L. cells were promoted without inhibition of cell growth. Received: 27 February 1997 / Revision received: 10 July 1997 / Accepted: 20 July 1997     

浙江富阳和海南文昌人工种植长春花生物碱积累组织特异性对比

药用植物长春花的人工栽培分布区主要在我国海南省,目前已引种扩大到浙江、四川等地。为了研究不同栽培区长春花生物碱积累的区域性差异和组织特异性分布特点,本文以浙江富阳和海南文昌两地所产的同苗龄长春花为原料,对其不同部位的长春碱、文朵灵和长春质碱的含量进行对比,分析3种生物碱在不同部位中积累的相关性。研究结果表明,海南文昌所产长春花植株的叶片中3种生物碱含量均高于浙江富阳。但分枝中长春碱含量特点是浙江富阳要显著高于海南文昌;不同部位生物碱之间相关性分析表明,叶片和主茎中文朵灵和长春质碱含量呈显著正相关,而分枝中长春碱与其前体长春质碱成极显著负相关。本文为长春花人工栽培区分布评估提供了理论指导。     

The negligible role of carbon dioxide and ethylene in ajmalicine production by Catharanthus roseus cell suspensions

Summary Removal of gaseous metabolites in an aerated fermenter affects ajmalicine production by Catharanthus roseus negatively. Therefore, the role of CO₂ and ethylene in ajmalicine production by C. roseus was investigated in 3 l fermenters (working volume 1.8 l) with recirculation of a large part of the exhaust air. Removal of CO₂, ethylene or both from the recirculation stream did not have an effect on ajmalicine production. Inhibition of ethylene biosynthesis in shake flasks with Co²⁺, Ni²⁺ or aminooxyacetic acid did not affect ajmalicine production. However, the removal of CO₂ did enhance the amount of extracellular ajmalicine.     

人工种植长春花生物学性状和生物碱含量的季节动态

以人工种植的长春花（Catharanthus roseus（L.）G. Don）为材料,研究了一个生长季节内长春花的生物学性状和生物碱含量的季节动态。研究发现,长春花的株高、生物量和叶片数呈现相似的生长趋势,即前期（5~7月）缓慢增长、中期（7~9月）快速增长和后期（9~10月）增长缓慢3个明显的季节生长特征,而花总数在前期和中期也呈现相似的规律,但在后期（9~10月）快速下降。长春花叶片中3 种生物碱含量的季节变化规律明显,变化趋势相一致。3 种生物碱的含量在一个生长季节内均是先增加后降低再增加的趋势,且3 种生物碱含量均在7月下旬和10月下旬出现2个明显的高峰值。长春花叶片中长春碱含量与长春质碱和文多灵含量之间均有较强的正相关,且与文多灵含量呈显著的正相关（p<0.05）,长春花叶片中长春质碱含量与文多灵含量之间呈显著的正相关（p<0.05）。因此,生产实践中人工种植长春花的最佳采收期是10月末,并可通过改变环境因子进一步诱导长春花叶片中长春碱类物质的积累。     

Improvement of indole alkaloid production in Catharanthus roseus cell cultures by osmotic shock

Catharanthine production in Catharanthus roseussuspension cell cultures was increased by about 4-fold to 28 mg l^–1, 23 mg l^–1and 24 mg l^–1by adding sodium alginate, mannitol or polyvinyl pyrrolidone, respectively. Sodium alginate and polyvinyl pyrrolidone also enhanced ajmalicine production to 28 mg l^–1and 31 mg l^–1, respectively. Up to 55–70% of the total alkaloids were released into the medium. These treatments could stimulate higher alkaloid production in C. roseuscell cultures than NaCl and KCl stresses. The possible mechanisms for these treatment effects are discussed.     

Effect of carbon and nitrogen growth limitation upon nutrient uptake and metabolism in batch cultures of Catharanthus roseus (L) G. Don

Cell suspension cultures of the Madagascan Periwinkle, Catharanthus roseus (L). G. Don were grown as batch cultures in two different types of media; in one medium the limiting nutrient was inorganic nitrogen, and in the other it was carbon. The response of the cells to these growth-limiting conditions was monitored by measuring cellular fresh weight, dry weight and protein accumulation, cell viability, medium sugar and nitrate levels, and the activities of certain intracellular enzymes throughout growth in batch culture. The enzymes investigated were glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), hexokinase (EC 2.7.1.40), phosphofructokinase (EC 2.7.1.11), nitrate reductase (EC 1.6.6.1), glutamate dehydrogenase (EC 1.4.1.2) and glutamine synthetase (EC 6.3.1.2). The effect of culturing the cells under different nutritional regimes was apparent in all aspects of growth; only some enzyme activities were unaffected. Cell viability remained at a high level for several days after growth limitation in both types of culture. The possibility that protein degradation in nitrogen-limited batch cultures is under very stringent control is discussed.