体外胁迫促进青蒿素在黄花蒿培养细胞中合成的研究

在黄花蒿细胞悬浮培养合成青蒿素的过程中添加微生物刺激剂和微生物胸外酶刺激剂,测定刺激剂对青蒿素合成的影响。采用的微生物刺激剂为霉菌ＡＳ３４,３０９和ＡＳ３,３４６、酵母Ｋluyberomyces fragilis及细菌ＡＳ１,３９８的提取物,微生物胸外酶刺激剂为果胶酶、纤维素酶及半纤维素酶。其中酵母Ｋluyberomyces fragilis提取物和果胶酶对青蒿素生物合成具有明显的刺激作用。用酵母Ｋluyberomyces fragilis提取物（２０％）处理黄花蒿培养细胞３天,青蒿素合成量达到每克黄花蒿干细胞３２０μｇ,比未刺激细胞的青蒿素合成量提高３８％;将果胶酶（０.２％）添加到青蒿素合成培养基中处理黄花蒿培养细胞２天以上,青蒿素合成量达到每克黄花蒿干细胞７４０μｇ,比未刺激细胞的青蒿素合成量提高３.０８倍。     

黄花蒿中青蒿素含量的动态变异性研究

目的研究黄花蒿生长发育过程中青蒿素含量的动态变异特性。方法分别在2、3、4、5、7月移栽黄花蒿苗,采集生长发育至各时期的黄花蒿,以紫外分光光度法,在292 nm处,对黄花蒿中青蒿素含量进行测定。结果黄花蒿下部叶中青蒿素含量明显高于上、中部;黄花蒿上午采收叶中青蒿素含量明显高于下午;在黄花蒿的整个生长发育过程中,从营养生长末期到花蕾期,青蒿素含量有递增趋势,开花后青蒿素含量明显下降;黄花蒿生长时间越长,叶中青蒿素的含量越高。结论黄花蒿应该选择在4月以前下种并移栽完成,选择在营养生长末期至花蕾期采收,采收的时间可选择在上午气温较低的晴天。     

我国黄花蒿中青蒿素含量的气候适宜性等级划分

针对黄花蒿种植中适生地选择的迫切要求,本文通过实地调查和查阅文献,获得全国各地青蒿素的含量数据。应用统计分析方法研究青蒿素含量与气候因子和地理分布之间的关系,应用ArcGIS软件的空间计算方法进行青蒿素含量的气候适宜性等级划分。结果显示:①青蒿素含量在我国各地差异较大,青蒿素含量纬向变异明显,北部高纬度地区青蒿素含量较低,南部青蒿素含量较高。北纬34度以南,东经(100～120°E)之间地区适宜黄花蒿的生长,青蒿素含量相对较高。北纬34度以北高纬度地区不适宜黄花蒿的生长,而且青蒿素含量低于0.2%。②在我国,亚热带湿润气候区最适宜黄花蒿的生长,而且青蒿素含量平均值大于0.5%。③温度、日照时数和降雨量是影响青蒿素含量高低的主要气候因子。最适宜青蒿素积累的气候条件为:温度(13.9～22℃)、日照时数(853～1 507 h)、降雨量(814～1 518 mm)。最适宜黄花蒿生长的气候条件为:温度(13～29℃)、降雨量(600～1 300 mm)。④广西西北部,四川、贵州、云南东部,重庆南部和湖南西部的气候条件最适宜黄花蒿的人工种植;湖北、安徽和江苏的南部地区也有适宜黄花蒿人工种植的气候条件。     

产青蒿素作物黄花蒿的研究现状及展望

黄花蒿（Artemisia annua L）是主产于我国的一种常见的草本植物,20世纪70年代其提取物青蒿素（Ar-temisinin）被发现。青蒿素在抗疟方面与传统的奎宁类抗疟药物具有不同的作用机理且疗效显著,近几十年来黄花蒿种植业在全国范围内得到了迅猛发展。随着对青蒿素研究的不断深入,如何最大限度的提高青蒿素产量,越来越成为人们研究的重中之重。由于目前人工合成青蒿素成本过高,不易进行批量生产,因此在当前阶段主要依靠人工种植黄花蒿来获取青蒿素,而地形,气候,黄花蒿的品种以及人工对黄花蒿生长的调控等都成为了左右青蒿素产量的重要因素。     

海南黄花蒿最佳采收期研究

目的 确定海南省黄花蒿的最佳采收期,为规范化种植黄花蒿提供依据.方法 采集不同生长期黄花蒿的叶或叶和花,计算其产量.采用超声波法提取青蒿素,紫外分光光度法测定其含量.结果 8月中旬海南黄花蒿中青蒿素的含量达到最高,为1.89%;而产量在9月初达到最大,平均单株产量可达56 g.结论 不同采收期黄花蒿中青蒿素的产量差异明显,海南黄花蒿最佳采收期为8月中旬.     

无载体固定化黄花蒿细胞生产青蒿素

来自黄花蒿的青蒿素具有极其明显的抗疟活性．成为临床首选的药物。因此青蒿素的获取成为关键。本研究采用无载体固定化法培养黄花蒿生产青蒿素，初步研究了无载体固定化细胞的生长特性．检测发现，利用该方法生产的青蒿素是常规细胞培养法的9倍，因此该方法有望成为青蒿素生产的首选方法。     

HPLC-UV-ELSD联用测定黄花蒿叶片中青蒿素及相关倍半萜的含量

目的HPLC-UV-ELSD联用建立黄花蒿叶片中青蒿素及其生合成相关倍半萜的含量测定方法。方法采用Venusil XBP-C18色谱柱(200 mm×4.6 mm,5μm),流动相:乙腈-水梯度洗脱,检测波长:203 nm,流速:1.0 mL.min-1,ELSD载气压力:0.35 MPa,喷嘴温度:35℃,漂移管温度:60℃,检测器:UV-ELSD联用,测定黄花蒿叶片中青蒿素、青蒿素B、3α-羟基-1-去氧青蒿素含量。同一色谱柱条件下流动相改为乙腈-水-甲酸(体积比为60.00∶40.00∶0.08),检测波长:215 nm,流速:1.0 mL.min-1,检测器:UV检测器,测定黄花蒿叶片中青蒿酸含量。结果4种倍半萜在各自的线性范围内线性关系良好(r≥0.9990),RSD<3%,平均加样回收率分别为98.5%(RSD=2.3%)、96.5%(RSD=1.8%)、97.5%(RSD=3.6%)、95.4%(RSD=2.5%)。结论该方法可作为黄花蒿中青蒿素及其生合成相关倍半萜的含量测定方法。     

重庆地区栽培黄花蒿中青蒿素的测定

目的 评价重庆不同产地黄花蒿样品质量,并为重庆市黄花蒿药材规模化发展建立方便快捷的含量测定方法。方法 采用紫外分光光度法,检测波长292 nm。结果 青蒿素对照品在0.02～0.14 mg&#8226;mL-1范围内具有良好的线性关系,r＝0.999 8（n＝7）,平均回收率为99.81%（n＝7）。重庆市云阳县等10份不同产地黄花蒿样品中青蒿素含量范围为6.71～10.19 mg&#8226;g-1。结论 重庆地区黄花蒿中青蒿素含量较高,具有一定的开发利用价值和工业生产价值。     

青蒿素生物合成的研究进展

青蒿素是上世纪70年代初中国科学家从药用植物黄花蒿(Artemisia annua L.)中分离得到的、含有过氧基团的新型倍半萜内酯抗疟药物。笔者综述了近年来青蒿素生物合成机理的研究进展,从青蒿素的前体如青蒿乙素和青蒿酸到青蒿素的合成,讨论了青蒿素生物合成的基本途径;从参与青蒿素合成的关键酶组成和功能,探讨了合成路线的主要构成,并介绍了青蒿素合成代谢调控的基因工程手段,为青蒿素的生物技术开发提供参考。     

两种培养基对黄花蒿挥发油体外抑菌活性的研究

目的利用普通营养琼脂培养基和M-H琼脂培养基测定黄花蒿挥发油的抑菌活性。方法采用滤纸片抑菌圈实验法测定黄花蒿挥发油抑菌效果,进而采用试管法测定黄花蒿挥发油的最低抑菌浓度(MIC)。结果黄花蒿挥发油对4种实验菌表现出不同的抑制活性,且黄花蒿挥发油在M-H培养基上抑菌活性优于普通培养基。     

Agrobacterium rhizogenes-mediated transformation of Artemisia annua: production of transgenic plants

Transgenic hairy roots were induced in the leaves of Artemisia annua by treatment with the LBA 9402 strain of Agrobacterium rhizogenes. The axenic hairy root cultures were found to produce the sesquiterpenes artemisinic acid and arteannuin B. The hairy root cultures were observed to spontaneously regenerate into plantlets on solid hormone-free MS medium. The regenerated plants had phenotypic characteristics typical to the transformed plants. Among the plants of the age of one month in culture, the transgenic plant was bigger (2.643 g/plant) than the normal (0.856 g/plant). Both these kinds of in vitro plants carried sesquiterpenes-artemisinic acid and arteannuin B.     

掌叶大黄毛状根培养及培养物中蒽醌类成分分析

大黄是我国特产的世界著名的传统中药，历代本草均有记载，始载于《神农本草经》，“味苦寒，有毒。主下瘀血、血闭、寒热，破征瘕积聚、留饮宿食，荡涤肠胃，推陈致新，通利水谷，调中化食，安和五脏”。作者已于近期报道了天山大黄发根的诱导方法［１］掌叶大黄Ｒｈｅｕ...     

Influence of Agrobacterium rhizogenes Strains on Biomass and Alkaloid Productivity in Hairy Root Lines of Hyoscyamus muticus and H. albus *

Using leaf explants of IN VITRO grown HYOSCYAMUS ALBUS and H. MUTICUS plantlets, hairy roots were induced following inoculation with AGROBACTERIUM RHIZOGENES strains A (4) and LBA-9402. The transformed roots, appearing after 14 - 17 days incubation on hormone-free MS medium containing 1 g/L cephalexin, were excised and maintained in the same medium. Ten randomly selected hairy root lines from each bacterial treatment of the two plant systems were compared for growth and alkaloid production in half-strength, hormone-free MS medium on 25 (th) day of culture. A. RHIZOGENES strain - A (4) induced hairy root lines of both H. ALBUS and H. MUTICUS were comparatively faster growing than those induced by strain LBA-9402. In contrast to earlier reports, some of the hairy root lines of H. ALBUS induced by A. RHIZOGENES strain A (4) were as fast growing as the hairy root lines of H. MUTICUS. The atropine yields of A (4) induced lines of H. ALBUS were significantly higher (3.5 fold) than the LBA-9402 induced lines. No such relationship between the bacterial strain and alkaloid productivity could, however, be obtained in case of hairy root lines of H. MUTICUS.     

Expression of Vitreoscilla hemoglobin enhances growth of Hyoscyamus muticus hairy root cultures

The Vitreoscilla hemoglobin gene (vhb) was introduced into Hyoscyamus muticus with the aim of investigating its effect on growth and alkaloid production of Agrobacterium rhizogenes-induced hairy root cultures. We were able to generate several VHb-expressing hairy root lines with different integration patterns. Substantial somaclonal variation was observed in growth and hyoscyamine production amongst both VHb-expressing lines and controls. Despite this variation, the growth properties of single lines remained stable over time. Expression of VHb was found to improve growth of H. muticus hairy roots in shake-flask cultures. The dry weights of the root cultures expressing Vitreoscilla hemoglobin were on average 18 % higher than those of the controls. VHb expression also increased the volumetric hyoscyamine production, mainly due to the improved growth properties. However, this difference was not statistically significant due to the wide somaclonal variation and fluctuations over time in both VHb and control hairy root lines.     

A study of the production of essential oils in chamomile hairy root cultures.

The active substances in chamomile (Matricaria recutita L.) belong to chemically different structural types. The largest group of medically important compounds forming the essential oils are primarily chamazulene, (-)-alpha-bisabolol, bisabololoxides, bisabolonoxide A, trans-beta-farnesene, alpha-farnesene, spathulenol and the cis/trans-en-in-dicycloethers. Flavonoids, coumarins, mucilages, mono- and oligosaccharides also have pharmacological effects. We studied the production of essential oils in genetically transformed cultures. Sterile juvenile chamomile plants were infected with A4-Y strains of Agrobacterium rhizogenes. They are known plant pathogens and are capable of inducing so-called hairy roots. The transfer DNA segment of the Ri-virulence plasmid of A. rhizogenes becomes integrated in the genome of the plant cells. The isolated hairy roots grow rapidly on hormone-free media. In order to obtain bacteria-free media, we cultured the transformed roots on Murashige-Skoog (MS) medium supplemented with carbenicillin (800 mg/l). To study the production of essential oils, the clones were propagated on liquid and solid MS and Gamborg (B5) media, respectively. According to gas chromatography, the composition of the essential oil of hairy root cultures on different media was found to be similar, but differing in proportion. The main component of the essential oil which was identified by gas chromatography and mass spectrometry was trans-beta-farnesene, as in the intact roots.     

Plant Nutritional Elements and Tropane Alkaloid Production in the Roots of Henbane (Hyoscyamus niger)

The effect of calcium on the morphology, growth, tropane alkaloid production (hyoscyamine and scopolamine) and plant nutritional element (calcium, magnesium and potassium) content of roots and root cultures of Hyoscyamus niger L. was examined in this study. It was observed that the tropane alkaloid productivity of root cultures was significantly higher than that of the corresponding field cultures. Murashige-Skoog (MS) medium was found to be optimal for the production of tropane alkaloids of the root cultures of H. niger. Variation in the calcium content of the medium had no substantial effect on the morphology, growth or content of magnesium, potassium and sodium of the root cultures of H. niger. However, increased calcium content in MS medium increased the production of the scopolamine and decreased the production of alkaloid hyoscyamine in the root cultures of H. niger. Collectively, our results are helpful in the optimization of medium composition for the production of highly valuable tropane alkaloid scopolamine using root cultures of H. niger.     

Production of Forskolin by Axenic Coleus forskohlii Roots Cultivated in Shake Flasks and 20-l Glass Jar Bioreactors*

Root cultures of COLEUS FORSKOHLII Briq. were initiated from primary callus or IBA-treated suspension cultures and maintained on Gamborg's B5 medium containing 1 mg/l IBA. Transformed root cultures were established by infecting surface-sterilized leaves with AGROBACTERIUM RHIZOGENES strain 15834. Transformation was confirmed by mannopine detection. These cultures displayed the typical characteristics of hairy root cultures, with the sole exceptions of slow growth in hormone-free medium and accelerated growth on medium containing phytohormones. All root cultures examined formed forskolin and its derivatives in amounts ranging from 500 to 1300 mg/kg dry weight, corresponding to about 4 to 5 mg/l. During cultivation roots could be cut into small pieces without affecting growth and forskolin production. Scale-ups of the cultivation procedure were performed in 20-l glass jars with a working volume of 10 to 13l. Forskolin production in bioreactors was better than in shake flasks. Levels of almost 14 mg/l could be reached after 21 d of cultivation. As in the shake flask experiments cutting the roots did not affect growth or productivity in a negative way.     

Tropane alkaloid production and shoot regeneration in hairy and adventitious root cultures of Duboisia myoporoides-D. leichhardtii hybrid

Co-culture conditions for Duboisia myoporoides-D. leichhardtii hybrid hairy root induction were investigated using leaf explants and Agrobacterium rhizogenes ATCC 15834. The bacteria density and duration of co-culture greatly affected the induction rate; the highest rate of 50% was obtained when the leaf explants were co-cultured for 2 d with 10(6) bacteria. One hairy root clone that showed the fastest root growth was selected and used for comparison study with adventitious roots cultured with 0.5 mg/l indole-3-acetic acid (IAA). The hairy roots cultured in Murashige and Skoog (MS) liquid medium grew well and yielded much more tropane alkaloids (35 mg/l scopolamine and 17 mg/l hyoscyamine) than adventitious roots cultured in 0.5 mg/l IAA after 6 weeks of culture at 25 degrees C in the dark. The hairy and adventitious roots (2.5 cm) grown in liquid media were divided into 5 parts (each 0.5 cm) along the root axis. Distribution of scopolamine and IAA was then determined by enzyme-linked immunosorbent assay (ELISA). Inverse relationship between contents of scopolamine and IAA was observed in the hairy roots; increase of scopolamine and decrease of IAA were proportional to the distance from the root meristem. In contrast, the contents of scopolamine and IAA were relatively constant in the adventitious roots. In shoot regeneration experiments, the hairy and adventitious root segments (1 cm) were placed onto 1/2 MS solid medium containing various concentrations of IAA and BA cultured at 25 degrees C under 16 h light. In adventitious roots, the shoots regenerated on media containing 6-benzyladenine (BA) (0.5 to 5 mg/l), and 100% regeneration was observed in medium with 0.1 mg/l IAA and 2 mg/l BA. On the other hand, shoot regeneration was only observed in 33% of hairy roots cultured on medium containing 5 mg/l BA.     

Polyacetylenes in hairy roots of a panax hybrid

From the hairy roots of an interspecific hybrid ginseng ( Panax ginseng x P. quinquefolium) known as Pgq, three polyacetylenes were isolated: panaxynol, panaxydol and 1,8-heptadecadiene-3,10-diol. These compounds isolated from the hairy roots were used for quantitative analysis to investigate the polyacetylene production of the hairy roots cultured in Gamborg B5 (B5) and 1/8 Murashige-Skoog (MS) liquid media. Maximum growth of the hairy roots was observed (ca. 5.4 g fresh weight/100 mL flask) at 8 weeks of culture in B5 medium. The highest total content of total polyacetylenes was 0.18 % of dry weight at week 8 when cultured in 1/8 MS medium. In addition, we compared the yields of polyacetylenes and ginsenosides in hairy roots cultured in B5 with those in 1/8 MS media and found the highest yields were obtained in the hairy roots cultured in B5 medium (1.24 mmol/flask polyacetylenes and 4.45 mmol/flask ginsenosides at week 8).     

Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids

Hairy roots, transformed with Agrobacterium rhizogenes, have been found to be suitable for the production of secondary metabolites because of their stable and high productivity in hormone-free culture conditions. A number of plant species including many medicinal plants have been successfully transformed with Agrobacterium rhizogenes. Transformed root cultures have also been found to be a potential source of high-value pharmaceuticals. In this article the most important alkaloids produced by hairy roots are summarised. Several different methods have been used to increase the alkaloid accumulation in hairy root cultures. The selection of high productive root lines based on somaclonal variation offers an interesting option to enhance the productivity. Elicitors and modification of culture conditions have been shown to increase the growth and the alkaloid production in some cases. Genetic engineering is a modern tool to regulate the secondary metabolism also in hairy roots. However, our knowledge on biosynthesis of many alkaloids is still poor. Only a limited number of enzymes and their respective genes which regulate the biosynthetic pathways are fully characterised.