Production of paclitaxel and baccatin III in a 20-L airlift bioreactor by a cell suspension of Taxus wallichiana

A cell suspension culture of Taxus wallichiana (Himalayan Yew) was grown in shake flasks and a 20-L airlift bioreactor running for 28 days in a batch mode, and its capacity to accumulate paclitaxel and baccatin III was measured. When both culture types were in the highest productive state (from day 24 to day 28), there was a greater accumulation of paclitaxel and baccatin III in the bioreactor culture than in the shake flask culture (factor of 2.0 and 1.2, respectively). These increases in paclitaxel and baccatin III production cannot be related to the difference observed between the growth rates of both cultures, because when the bioreactor culture was at maximum productivity, its cell biomass, expressed in g L(-1) of dry weight, was similar to that obtained in the shake flask culture. It seems that these improvements were mainly due to adequate aeration and mixing of the culture in the bioreactor. The maximum yield observed for paclitaxel (20.84 mg x L(-1) day 24) and baccatin III (25.67 mg x L(-1) day 28) represents a productivity of 0.90 mg x L(-1) d(-1) and 0.93 mg x L(-1) x d(-1) respectively.     

Production of triterpenoids in liquid-cultivated hairy roots of Galphimia glauca

The production of three triterpenoids from Galphimia glauca hairy root cultures, the sedative principle galphimine E (2), the recently described glaucacetalin A (3), and maslinic acid (6), was quantified by HPLC in the biomass and the culture medium. Batch cultures of the hairy root line VYT, obtained through infecting cotyledons with Agrobacterium rhizogenes ATCC 15 834, were grown for 41 days in shake flasks containing B5 medium without phytohormones. A maximum biomass of 11 g/L DW was obtained on day 33, while the doubling time was 6 days. Throughout the growth cycle fresh and dry weights as well as triterpene production were registered. Glaucacetalin A (3), excreted into the culture media, reached a maximum amount of 2.14 mg/L after 21 days while galphimine E (2) and maslinic acid (6) were recovered from the root biomasses reaching maximum concentrations of 0.11 and 0.43 mg/g, respectively, on day 39.     

Auxins affected ginsenoside production and growth of hairy roots in Panax hybrid

Hairy roots of interspecific hybrid ginseng (Panax ginseng x P. quinquefolium), induced by Agrobacterium rhizogenes ATCC 15834, grew well in B5 liquid media supplemented with 2.5 microM auxins (3-indole butyric acid (IBA), 1-naphtaleneacetic acid (NAA) and 3-indoleacetic acid (IAA)). The hairy roots cultured in B5 liquid medium supplemented with 2.5 microM IBA showed best growth (6.39 g fresh weight per a flask, at week 8). The highest content of the total ginsenosides was 1.63% as dry weight at week 8 when cultured with 2.5 microM NAA. The different auxins affected the numbers and lateral branching roots. Especially, 2.5 microM IBA promoted the lateral root formation (43.7+/-4.0 roots, at week 8), and 2.5 microM NAA promoted the lateral root growth (45.3+/-5.6 mm, at week 8). The growth and ginsenosides production of 8-week old hairy roots cultured in B5 liquid media supplemented with IBA and NAA combinations were also investigated. Hairy roots produced higher amounts of ginsenosides in B5 liquid media supplemented with 0.5-1.0 microM IBA and NAA combinations than that cultured in B5 liquid media supplemented with only IBA and NAA. The highest yield of ginsenoside was obtained when cultured with 0.5 microM IBA and 1.0 microM IBA combination (6.38 mg per a flask, at week 8).     

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...     

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).     

Enhancement of tanshinone production in Salvia miltiorrhiza hairy root culture by Ag+ elicitation and nutrient feeding

Ag (+) was employed as an abiotic elicitor to stimulate the secondary metabolite production in hairy root culture of Salvia miltiorrhiza, a valuable herbal plant. The addition of Ag (2)S (2)O (3) at 15 - 40 microM to the culture between 12 and 22 days post inoculation resulted in more than 2-fold increase in the yields of the three diterpenoid tanshinones, tanshinone I, tanshinone IIA and cryptotanshinone. This stimulating effect was dependent on the Ag (+) dose, the day of Ag (+) addition to the culture and the tanshinone species, while the total yield of the three tanshinones (TT) was mainly dependent on the Ag (+) dose, with the highest being attained at 30 microM. The Ag (+) treatment also caused a dose-dependent inhibition of hairy root growth. Sucrose feeding or medium renewal before the addition of Ag (+) to the culture effectively prevented the growth inhibition, and significantly increased the biomass concentration and volumetric tanshinone yield. With combined medium renewal and Ag (+) treatment, in particular, the TT yield was increased 6.6-fold relative to that of the control (55.7 mg/L versus 7.3 mg/L). The elicitor function of Ag (+) in the hairy root culture was validated by its ability to induce the characteristic elicitor responses of plants, the increase in cross-cell membrane ion fluxes and the production of reactive oxygen species.     

Apoptotic mechanisms of the biotechnologically produced arylnaphtalene lignan justicidin B in the acute myeloid leukemia-derived cell line HL-60

BackgroundThe present study aimed at optimization of the biotechnological production of the lignan justicidin B by genetically transformed cultures of Linum leonii and the pharmacological evaluation of the pro-apoptotic effects of the compound in HL-60 cells.MethodsA rapidly growing selected root line of L. leonii was grown in 2-L bioreactor for period of 40 days and the protocols for obtaining of the compound have been optimized. The pharmacological study included evaluation of the cytotoxicity of the compound in HL-60 cells (MTT-assay), its apoptogenic effects and its effects on caspase 3,8 and 9 activation.ResultsAfter 40 days of sterile run scale up of hairy root culture in bioreactor, 27.2 g/L dry weight of root biomass was harvested from the bioreactor culture vessel, recording about nine times increase over initial inoculum (3.0 g), with 1.55% ± 0.07 Justicidin B, greater than yields from 300 ml flasks. Our findings are the first work toward the scale up of L. leonii hairy roots-based biotechnological production of Justicidin B, employing bioreactors for high biomass production to meet the industrial requirement. The results from the pharmacological evaluation have shown that the tested arylnaphtalene lignan is a potent cytotoxic and proapoptotic agent against HL-60. The induction of apoptosis proceeds via activation of the intrinsic mitochondrial cell-death signaling pathways.ConclusionThe potent activity at low micromolar concentration and the feasibility of biotechnological production of justicidin B implies that there is enormous scope in its further evaluation as possible antineoplastic drug candidate.     

Immobilization of Galphimia glauca plant cell suspensions for the production of enhanced amounts of Galphimine-B

We tested the capacity of Galphimia glauca cells to produce galphimine-B (G-B) when under the effects of a two-stage culture system: cell immobilization in Ca2+-alginate beads and culture scale-up from shake-flask to two different types of bioreactor (stirred and airlift). In the shake-flask culture, using optimum media for cell growth (first stage) and G-B production (second stage), the G-B yield was similar in both immobilised and free cells. However, while the free cells accumulated G-B within cytoplasmatic compartments, where it could not be recovered without cell disruption, immobilized cells excreted up to 100 % of the G-B produced. Immobilized cells grown in bioreactors running for 14 days with growth medium and an additional 26 days with production medium in batch mode showed a high G-B yield. The stirred bioreactor was the most efficient with a G-B content in the culture medium of 1381 microg.L (-1) at day 24 of culture.     

基于中国仓鼠卵巢细胞表达的Fc融合蛋白制备工艺的优化

目的 优化基于中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞表达的Fc融合蛋白制备工艺.方法 对于上游细胞培养工艺,先在摇瓶中分别对培养基配比和流加培养方案进行优化,再将优化工艺放大到生物反应器规模并确认其可行性.对于下游蛋白纯化工艺,分别对A蛋白亲和层析的洗脱条件和阴离子交换层析的平衡条件进行优化.结果 对于上游细胞培养工艺,以种子培养基∶基础培养基(CD OptiCHO)∶基础培养基(CDM4Mab)为2∶1∶1的配比配制培养基,并以3、6和9d补料的流加培养方案进行细胞培养,可获得理想的细胞密度和活率,且成本较低.对于下游蛋白纯化工艺,以pH3.3的洗脱液进行A蛋白亲和层析纯化时,Fc融合蛋白的回收率为94.7％,纯度为98.64％;以pH5.0～pH5.5的平衡液进行阴离子交换层析纯化时,Fc融合蛋白的回收率达到98％以上、纯度达到99％.结论 基于CHO细胞表达的Fc融合蛋白制备工艺的上游细胞培养和下游蛋白纯化工艺得到成功优化,这为此类抗体药物制备工艺的优化提供了思路.     

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.     

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.     

Datura metel: in vitro production of tropane alkaloids

Hairy root lines of Datura metel were established following infection of aseptic stem segments with Agrobacterium rhizogenes strain A4 and cultured in hormone-free B5 solid medium. The growth and production of hyoscyamine and scopolamine (mg/g dry wt.) of these root cultures was encouraged by using B5 liquid medium with half-strength salts. In these culture conditions, the capacity of the highest productive root line 25 to excrete scopolamine into the culture medium rose from 8.7% to 70% when the permeabilizing agent Tween 20 was added for 24 h to the medium, after 2 and 4 weeks of culture. Using an airlift bioreactor (41) with modifications in order to increase root anchorage, the Tween 20 treatment encouraged both growth and alkaloid productivity of cultured root line 25. After 4 weeks their biomass yield was 2.3 mg/l/day and 0.84 mg/l/day of scopolamine was produced (70% extracellular). The scopolamine released into the culture medium was separated with an Amberlite XAD-2 column located in the media exit.     

黄花蒿发根的液体培养及青蒿素合成的动态特征研究

目的:探讨黄花蒿发根生长及青蒿素生物合成的动态特征,建立高效、稳定的黄花蒿发根液体培养体系。方法:测定不同培养基以及MS培养基中不同营养元素对黄花蒿发根生物量和青蒿素含量的影响。结果:筛选出优化的黄花蒿发根液体培养基,获得拟合的黄花蒿发根生长和青蒿素合成的Logistic方程。结论:在优化的MS培养基中黄花蒿发根生长迅速且能稳定合成青蒿素,为工业化大规模生产青蒿素提供了可能。     

葡萄糖和Cu2+对蛹虫草Cu,Zn超氧化物歧化酶在E.coli中表达的影响

对含有蛹虫草Cu，Zn-超氧化物歧化酶（cm—SOD）基因的重组E．coli培养条件进行了初步优化。结果表明，LB培养基中含有Cu^2＋可明显提高超氧化物歧化酶比活力；而在菌体培养中补加葡萄糖，可提高重组蛋白表达量和细胞生物量。在含有0．6mmol／L Cu^2＋、Zn^2＋的LB培养基中补加22mmol／L葡萄糖，每200ml培养液可获得1g湿细胞，超氧化物歧化酶比活力达到3305u／mg。     

Ginsenoside Rg3 mediates endothelium-dependent relaxation in response to ginsenosides in rat aorta: role of K+ channels

The aim of the present study was to characterize the endothelium-dependent relaxation elicited by ginsenosides, a mixture of saponin extracted from Panax ginseng, in isolated rat aorta. Relaxations elicited by ginsenosides were mimicked by ginsenoside Rg1 and ginsenoside Rg1, two major ginsenosides of the protopanaxatriol group. Ginsenoside Rg3 was about 100-fold more potent than ginsenoside Rg1. The endothelium-dependent relaxation in response to ginsenoside Rg3 was associated with the formation of cycle GMP. These effects were abolished by N(G)-nitro-L-arginine and methylene blue. Relaxations in response to ginsenoside Rg3 were unaffected by atropine, diphenhydramine, [D-Pro2, D-Trp7,9]substance P, propranolol, nifedipine, verapamil and glibenclamide but were markedly reduced by tetraethylammonium. Tetraethylammonium modestly reduced the relaxation induced by sodium nitroprusside. These findings indicate that ginsenoside Rg3 is a major mediator of the endothelium-dependent nitric oxide-mediated relaxation in response to ginsenosides in isolated rat aorta, possibly via activation of tetraethylammonium-sensitive K+ channels.     

响应曲面法优化人参总皂苷动态连续循环提取工艺

目的优化人参总皂苷的提取工艺。方法在单因素试验考察乙醇体积分数、药材粒径、溶剂流速、浓缩温度对人参总皂苷转移率的影响基础上,通过响应曲面法对人参总皂苷动态连续循环提取工艺进行优化。结果人参总皂苷的含量与所考察3个因素之间关系符合二次回归模型。优化后人参总皂苷的最佳提取工艺为,乙醇体积分数78%,超微粉碎时间10min,溶剂流速24mL/min,浓缩温度65℃。人参总皂苷转移率为85.64%(P<0.0001),验证试验结果与预测值偏差为2.58%,说明该模型比较可靠。结论该方法科学、合理、可行,对人参总皂苷提取工艺应用于实际生产具有重要的参考价值。     

Production of amarogentin in root cultures of Swertia chirata

Conventional and Agrobacterium rhizogenes-transformed root cultures were studied with respect to growth and amarogentin content following cultivation in various growth media. The fastest growth rate was observed using Nitsch medium. The best amarogentin content was obtained after cultivation in root culture (RC) medium for which the slowest growth rate was noticed. Addition of sucrose at 6% and 9% (w/v), respectively, also resulted in better growth rates and increased total but unaltered relative amarogentin content compared to 3% (w/v) sucrose. No change in amarogentin content was observed upon addition of elicitors, putative precursors of amarogentin biosynthesis, and plant growth hormones with the exception of salicylic acid and chitosan: at 100 mM salicylic acid a reduction and at 25 mg/L chitosan an increase of amarogentin were observed at significant levels. The cultivation of S. chirata roots in a 2-L stirred-tank bioreactor was successful only with a stainless-steel mesh fitted inside the culture vessel for immobilization of the roots. A 15-fold enhancement of amarogentin content in the medium was achieved by a root permeabilisation treatment using Tween 20 at 1.3% (v/v) final concentration in the bioreactor.     

巴斯德毕赤酵母表达人微小纤溶酶原的放大研究

目的:研究巴斯德毕赤酵母(Pichia pastoris)表达重组人微小纤溶酶原(rh-mPlg)的放大工艺。方法:分别采用1 L摇瓶、7.5 L发酵罐对Pichia pastoris工程菌rh-MPLG/GS115进行高密度培养、甲醇诱导表达。摇瓶培液经2步纯化:SP-Seph-arose FF、Superdex 75;发酵罐培液经3步纯化:超滤、Sephacryl S-100、Q-Sepharose FF,活性组分透析后冷冻干燥。以组织型纤溶酶原激活剂(t-PA)激活rh-mPlg,纤维蛋白平板测定其纤维蛋白溶解活性,计算并比较2种制备方法所获rh-mPlg比活性及得率。结果:采用1 L摇瓶和7.5 L发酵罐发酵可分别获得34 mg.L-1培液、210 mg.L-1培液的得率,经纯化后rh-mPlg纯度分别为95%和97%,比活性分别为20.6和23.0 U.mg-1。结论:以发酵罐生产rh-mPlg,可显著提高其产量,且比活性与摇瓶表达相近,验证了放大生产的可行性。     

人参皂甙对小脑生长锥影响的体外培养观察

自吉林人参根部制备的总皂甙(Rx),按每毫升培养液含50,100,200μg的浓度,对新生(P-O)B_6C_3小鼠的小脑进行Maximow器官——组织型培养,总数达120片。在体外培养第2,3,5天分别在相差显微镜下进行活体观察,对生长锥的长度和密度作进一步的观察和测量,同时结合神经染色和扫描电镜的观察与记录,结果显示实验组在生长锥的生长趋势较对照组明显,尤以100μg/ml组最为显著,具有统计学意义。本研究结果提示人参皂甙对神经组织的生长和神经网络的建立具有促进作用。     

地衣芽孢杆lw-72合成纤溶酶的摇瓶发酵工艺优化

通过单因素试验和正交试验确定地农芽孢杆菌lw-72摇瓶发酵合成纤溶酶的最佳条件,最佳培养基组成为:玉米粉0.5%、黄豆饼粉2.0%、Na_2HPO_4 0.4%、NaH_2PO_4 0.2%、CaCl_2·2H_2O 0.01%、KCI 0.01%;培养摹初始pH为7.0;最佳培养条件:装液量30 ml/250 ml摇瓶,接种量10%,发酵时间为72 h.在该条件下发酵液中纤溶酶酶活力为622 u/ml,是初始发酵培养基的3.4倍.