固定化培养和产物释放促进剂对硬紫草细胞代谢的影响

固定化培养的硬紫草细胞生长缓慢,仅包理球外层的细胞生长明显,其蛋白质合成的量也低。培养３０ｄ的细胞色素产量达到４．２ｍｇ／ｇＦＷ,相对色素分泌量达到７０％,而色素的组成成分及各组分的比例也与悬浮细胞的不同。以正十六烷处理固定化细胞可促进产物释放,其不同的处理时间对细胞没有显著影响。连续培养的固定细胞保持其色素形成能力达８０ｄ之久,色素总产量达２０ｍｇ／ｇＦＷ．     

固定化培养和产物释放促进剂对硬紫草细胞代谢的影响

固定化培养的硬紫草细胞生长缓慢,仅包埋球外层的细胞生长明显。其蛋白质合成的量也低。培养３０ｄ的细胞色素产量达到４．２ｍｇ／ｇＦＷ,相对色素分泌量达到７０％,而色素的组成成分及各组分的比例也与悬浮细胞的不同。以正十六烷处理固定化细胞可促进产物释放,其不同的处理时间对细胞没有显著影响。连续培养的固定细胞保持其色素形成能力达８０ｄ之久,色素总产量达２０ｍｇ／ｇＦＷ。     

大孔吸附树脂吸附分离紫草色素的研究

研究了几种大孔吸附树脂对紫草色素的吸附提取。结果表明,NKA-Ⅱ具有较高的吸附量,且易于解吸,适于在新疆紫草细胞大量培养过程中用于对紫草色素的吸附分离。     

新疆紫草细胞悬浮培养过程研究

新疆紫草细胞生长和紫草素合成之间属非生长偶联型,所以采用二步培养法研究悬浮培养过程。新疆紫草细胞悬浮培养的生长周期约为21 d,紫草素合成周期约为16 d。新疆紫草细胞生长阶段培养液的电导率与生物量呈线性负相关,随着生物量的增加,培养液的电导率降低。因此,可以通过测量电导率来预测培养体系中生物量的变化情况。细胞生长过程中硝酸盐、铵盐和可溶性糖的消耗与生物量的变化具有很好的线性相关性,基于硝酸盐、铵盐和可溶性糖的细胞收率系数分别为8.64、104.3和0.68 g/g。     

硬紫草细胞悬浮培养和紫草宁及其衍生物的形成

硬紫草细胞悬浮培养形成紫草宁及其衍生物时．细胞生长曲线呈扁平的s形。细胞停止生长后,紫草宁及其衍生物大量形成,二者的动态变化呈负相关。测定丁此过程中培养液的无机元素和可溶性糖含量、溶氧、pH值以及细胞形态的变化情况,可作为硬紫草细胞大规模培养的参考。     

细胞固定化载体比表面的测定

为了衡量细胞固定化载体的性能。基于单分子层吸附理论,利用溶液中亚甲蓝染料在固形物表面的吸附倾向;建立了用于测定细胞固定化载体比表面的“动态染料吸附法”,方法学考察时以PVA-海藻酸钠的混合载体为例,结果表明四批次测量同一载体比表面的结果变异系数为5.5%,测量的载体比表面能精确反映载体内PVA,或是海藻酸钠浓度的变化,说明方法重复性好,灵敏度高,同时讨论了文献中“染料吸附法”测定比表面的不足。     

紫草的制剂学研究进展

本文对国内有关紫草制剂的研究概况进行了综述,为研究和开发紫草制剂提供依据.     

紫草提取物的体外抗氧化活性研究

为研究紫草色素(LEP)的体外抗氧化活性,采用常规回流方法提取LEP,测定了LEP对超氧自由基(O2)和1,1-二苯基-2-苦苯肼自由基(DPPH.)的清除能力,及其对β-胡萝卜素/亚油酸自氧化体系的抑制作用。结果表明,LEP对DPPH.和O2均有较强的清除能力,并且对β-胡萝卜素/亚油酸自氧化体系有明显的抑制作用,说明紫草的药理作用可能与LEP较强的抗氧化能力有关。     

根癌农杆菌转化紫草的研究

紫草 (ＬｉｔｈｏｓｐｅｒｍｕｍｅｒｙｔｈｒｏｒｈｉｚｏｎＳｉｅｂ .ｅｔＺｕｃｃ)是传统中药。其根部含有萘醌类化合物—紫草素及其衍生物 ,具有显著的抗菌、抗炎、抗癌以及促进伤口愈合等生理活性。紫草素同时也是一种名贵化妆品染料。科学家对紫草的研究兴趣是基于其资源的缺乏及紫草植物本身所具有的一些特点 ;如 :紫草素及其衍生物的颜色特性可凭借肉眼观察 ,紫草素及其衍生物只在紫草的根部积累 ,紫草素合成的次生代谢途径受多种酶和外界条件 (光照 ,营养等 )的调节等。紫草细胞培养 (Ｆｕｊｉｔａ等 ,1983;叶和春等 ,1991)可以产…     

Shikonin derivative formation on the stem of cultured shoots in Lithospermum erythrorhizon

Lithospermum erythrorhizon , which are capable of producing red pigments, have been established. The red pigments were formed on the stems of L. erythrorhizon shoots cultured both on solid and in liquid media without phytohormones at 25 °C in the dark. Thin-layer chromatography, high-performance liquid chromatography and ¹ H nuclear magnetic resonance analyses revealed that the red pigments which accumulated on the cultured shoots were shikonin derivatives. The effects of various basal media and phytohormones (indole-3-acetic acid, indole-3-butyric acid and kinetin) on the growth and the formation of shikonin derivatives were investigated. When the shoots were cultured on Murashige and Skoog solid medium, the addition of kinetin remarkably enhanced shikonin derivative accumulation in the shoots. However, these effects of kinetin were not observed in the liquid culture when cultured in Gamborg B5 medium. The maximum content of shikonin derivatives (2.3% as dry weight, ca. 1.5 mg/100 ml flask) was observed in the shoots cultured in phytohormone-free B5 liquid medium for 5 weeks. Received: 1 February 2000 / Revision received: 23 March 2000 / Accepted: 28 March 2000     

硬紫草悬浮细胞原生质体培养形成愈伤组织及其体细胞克隆变异

从硬紫草悬浮细胞系ＡＲ１２６分离原生质体,只有用葡萄糖作渗透剂经琼脂糖－液体双层培养才能获得可见的原生质体克隆,从中选择３４个克隆,用两阶段法生产紫草素及其衍生物,测量了它们在两阶段的细胞生长量及生产阶段的色素含量,并比较了其分布规律,其中最好的原生质体克隆色素生产量是起始悬浮系的２．５４倍,经培养４０ｄ达４４．０６ｍｇｇ－１ＦＷ,而且其生产量在所测的８０ｄ内无明显下降。     

Enzyme activity and shikonin production in Lithospermum erythrorhizon cell cultures

The activities of the biosynthetic enzmes phenylalanine ammonia lyase (PAL) and 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) were measured in cells transferred from growth to production medium in a two-stage batch culture. It was found that both these enzymes showed transient increases, PAL (three- to fourfold) and HMGR (two- to four-fold), at or near the point of exhaustion of nitrogen source (NO(3)). Production of shikonin derivatives also started at this time. The addition of excess nitrate to the medium shortly before nitrate exhaustion (days 6-8) markedly reduced the final product yield (by 70-80%) while addition of excess nitrate in the later stationary growth phase (days 14-16) had no significant effect. When the production rate of shikonin derivatives was correlated with PAL activity, it was observed that production rate is very low (less than 1 mg/L . day) at low levels of PAL activity (below 0.1 unit/mg protein). Once a threshold level of PAL activity (about 0.15 unit/mg protein) is reached, the biosynthetic rate of shikonin derivatives increases. Such a relationship could not be deduced for HMGR activity. It was concluded that the production of shikonin derivatives may be limited at the phenylalanine deaminating step at low levels of PAL activity.     

Exploring the evolutionary process of alkannin/shikonin O-acyltransferases by a reliable Lithospermum erythrorhizon genome

Increasing genome data are coming out. Genome size estimation plays an essential role in guiding genome assembly. Several months ago, other researchers were the first to publish a draft genome of the red gromwell (i.e. Lithospermum erythrorhizon). However, we considered that the genome size they estimated and assembled was incorrect. This study meticulously estimated the L. erythrorhizon genome size to should be ∼708.74 Mb and further provided a reliable genome version (size ≈ 693.34 Mb; contig_N50 length ≈ 238.08 Kb) to support our objection. Furthermore, according to our genome, we identified a gene family of the alkannin/shikonin O-acyltransferases (i.e. AAT/SAT) that catalysed enantiomer-specific acylations in the alkannin/shikonin biosynthesis (a characteristic metabolic pathway in L. erythrorhizon’s roots) and further explored its evolutionary process. The results indicated that the existing AAT/SAT were not generated from only one round of gene duplication but three rounds; after different rounds of gene duplication, the existing AAT/SAT and their recent ancestors were under positive selection at different amino acid sites. These suggested that a combined power from gene duplication plus positive selection plausibly propelled AAT/SAT’s functional differentiation in evolution.