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。     

紫草素对人肝癌HepG2细胞增殖的抑制及诱导凋亡作用

目的:观察紫草素抑制人肝癌HepG2细胞增殖及凋亡诱导的作用。方法:用不同浓度的紫草素处理HepG2细胞,MTT检测紫草素对HepG2细胞生长增殖的抑制作用;比色法测定Caspase-3酶活性;Western blot法检测磷酸化Akt蛋白(pAkt)的表达。结果:紫草素能够抑制人肝癌HepG2细胞的增殖,并呈浓度、时间依赖性,紫草素与HepG2细胞作用24小时后Caspase-3酶活性显著增强,显示紫草素诱导的调亡作用随时间的延长而增加;同时,紫草素处理HepG2细胞后,随着药物浓度的增加,磷酸化Akt蛋白表达下降。结论:紫草素可抑制人肝癌细胞HepG2的增殖,诱导HepG2细胞凋亡,凋亡机制可能与紫草素抑制PI3K/Akt信号途径有关。     

真菌诱导子对新疆紫草悬浮培养细胞的生长和紫草素合成的影响

４种真菌诱导子诱导新疆紫草悬浮培养细胞对细胞紫草素的合成均有促进作用,其中以黑曲霉诱导子效果最高,而且促进细胞紫草素的外排,约３０％的紫草素存在于培养液中;诱导促使细胞苯丙氨酸解氨酶活性显著提高,培养液中紫草素的含量变化与苯丙氨酸解氨酶活性变化呈正相关性。     

密花滇紫草萘醌成分研究及紫草素含量测定

从密花滇紫草(Onosma confertum W.W.Smith)根的石油醚提取物中分离出四种化合物,根据其理化常数、光谱数据(MS、IR、~4H-NMR、CD)及 HPLC 等鉴定为:去氧紫草素(Ⅰ)、β,β-二甲基丙烯酰紫草素(Ⅱ)、乙酰紫草素(Ⅲ)和紫草素(Ⅳ)。并对紫草素衍生物碱水解所得紫草素总含量进行了测定。     

滇紫草愈伤组织培养与紫草素产生

浓度为10~(-5)Smol/1和10~(-6)mol/l的2,4-D和NAA分别与10~(-5)mol/l的KT组合,能明显抑制滇紫草(Onosma paniculatum Bur. et Fr.)愈伤组织中紫草素的产生,但几乎不受天然生长素IAA和KT组合的影响。葡萄糖较蔗糖能更有效地促进紫草素的产生,它们的最适浓度均为6％。LH和CH能抑制紫草素的产生,CH浓度大于0.02％时能抑制愈伤组织的生长,LH对生长无明显影响。椰乳浓度为10％时,能明显地促进紫草素的产生,紫草素的含量是对照的24倍。     

真菌诱导子对新疆紫草悬浮培养细胞的生长和紫草素合成的影响

4种真菌诱导子诱导新疆紫草悬浮培养细胞对细胞紫草素的合成均有促进作用,其中以黑曲霉诱导子效果最高,而且促进细胞紫草素的外排,约30％的紫草素存在于培养液中;诱导促使细胞苯丙氨酸解氨酶活性显著提高,培养液中紫草素的含量变化与苯丙氨酸解氨酶活性变化呈正相关性。     

多糖对滇紫草培养细胞的影响

黑节草多糖、火棘果果胶和琼脂加入到培养基中,均能促进滇紫草细胞合成紫草素。黑节草多糖促进紫草素合成的最适浓度为0.05—0.1％,大于最适浓度紫草素合成逐渐受到抑制。三种黑节草多糖单体中以多糖Ⅱ最为有效,紫草素产量为90.07mg/l。加入火棘果果胶和琼脂均对紫草素合成呈现促进作用。最后对多糖的作用机理以及多糖与寡糖的关系进行了讨论。     

紫草及其提取物生物学活性研究进展

紫草在我国有悠久的药用历史,其味甘,咸,性寒,归心包络,肝经,有凉血、活血、清热、解毒等功效,中医临床主要用于湿性斑疹、紫癜、血尿、淋浊和血痢、热结便秘、烧伤、湿疹、丹毒、痈疡等病。2005版《中国药典》收录紫草2个品种,分别是紫草科新疆紫草[(Arnebia euchroma(Royle)Johnst]、内蒙紫草(Arnebia gutlata Bunge)的干燥根[1]。已从紫草中分离提取出多种具有生物学的活性成分,其中紫草根中主要含萘醌类成分和脂肪酸成分,萘醌类成分主要有紫草素(Sh ikon in)、乙酰紫草素(Acetylsh ikon in)、去氧紫草素(Deoxysh ikon in)、异丁酰紫…     

寡糖素对滇紫草愈伤组织色素合成的影响

从黑节草、红花和人参中提取的寡糖素对滇紫草愈伤组织的色素合成均能起促进作用,当它们单独使用时,分别找到了它们作用于愈伤组织以生产色素的最适浓度.本研究对将来采用组织培养方法进一步工业化生产紫草素提供了依据。     

紫草素及其衍生物的调控策略

紫草素及其衍生物在医药、食品、化妆品和印染等领域有着巨大的市场潜力,如何提高它们的产量已成为该领域研究的热点。综述了调控紫草素及其衍生物的合成和积累方法,主要包括高产细胞系的筛选、生产培养基的改良、外加物和外加条件的影响、基因工程调控、生物反应器的影响及分析和提取纯化技术等,并对有关紫草素及其衍生物今后的发展方向进行了展望。     

滇紫草（Onosma paniculatum） 愈伤组织中主要成分的抑菌作用

从诱导形成的滇紫草愈伤组织中分离得到四种单体[3]．其中两种单体:β、β-二甲基丙烯酰阿卡宁（β,β-dimethyacrylalkannin）和乙酰阿卡宁(Acetylalkannin)的愈伤组织混合色素,对金黄色葡萄球菌和假单孢杆菌都有比较明显的抑制作用。尤其是乙酰阿卡宁抑制效果格外显著。然而对大肠杆菌却表现不出抑制效应。     

Purple-red Pigment Formed in Callus of Onosma paniculatum Bur.et French

The callus of Onosma paniculatum produced by young roots and stems with 2-staged culture method contains slightly higher contents of the purple-red pigment than the original plant. This pigment is a naphthoquinone compound consisting of six shikonin derivatives, whose Rf values are very close to those of shikonin derivatives in the intact root and stem. Four monomers of shikonin have been obtained with the columned chromatography of silica gel H from the callus. The Structure analysis shows that the shikonin derivatives are deoxyshikonin, β, β-dimethy- lacrylalkannin, acetylalkannin and β-acetoxyisovalerylalkannin.     

Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO₄) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.     

Genetic engineering on shikonin biosynthesis: expression of the bacterial ubiA gene in Lithospermum erythrorhizon

The naphthoquinone pigment shikonin from Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) was the first plant secondary metabolite produced in industrial scale from plant cell cultures. We have now manipulated the biosynthetic pathway leading to shikonin in L. erythrorhizon by introduction of the bacterial gene ubiA. This gene of Escherichia coli encodes 4-hydroxybenzoate-3-polyprenyltransferase, a membrane-bound enzyme that catalyzes a key step in ubiquinone biosynthesis. Using geranyl diphosphate (GPP) as substrate, it is able to catalyze the formation of 3-geranyl-4-hydroxybenzoate (GBA), a principal step of shikonin biosynthesis. The prokaryotic ubiA gene was fused to two signal sequences for targeting of the resulting peptide to the endoplasmic reticulum (ER). Constructs with different constitutive promoters were introduced into L. erythrorhizon using Agrobacterium rhizogenes-mediated transformation. In the resulting hairy root lines, high UbiA enzyme activities could be observed, reaching 133 pkat mg(-1). Expression of ubiA resulted in an accumulation of GBA in an amount exceeding that of the control culture by a factor of 50. However, the ubiA-transformed lines showed only a marginal (average 22%) increase of shikonin production in comparison to the control lines, and there was no significant correlation of UbiA enzyme activity and shikonin accumulation. This suggests that overexpression of ubiA alone is not sufficient to increase shikonin formation, and that further enzymes are involved in the regulation of this pathway.     

Fractionation and Biological Activity of Aspergillus oryzae Elicitor Promoting Biosynthesis of Shikonin Derivatives

The Aspergillus oryzae elicitor was extracted from mycelia. The concentrated crude preparation of which was treated through DEAE-Cellulose, Sepharose-4B. Bio-Gel p- 4 and 732 columns. Elicitor activity was associated with fraction F Ⅰ b2-H, which had no affinity for DEAE-Cellulose and 732 resin. Its molecular weight was 1200～2200 D and its carbohydrate content was 6.7% of that of the crude. The elicitor activity was 120 times higher than that of crude preparation. There were also fractions F Ⅱ of nucleic acids and F Ⅰ b2-Na of nucleotides, amino acids in crude elicitor preparation. They did not affect shikonin derivative formation at low concentration, but inhibited shikonin derivative formation at high concentration. Fraction FIa of polysaccharid nature in the crude preparation which strongly inhibited shikonin derivative formation was another kind of elicitor of a new metabolite yellow pigment.     

Enhanced shikonin production from Lithospermum erythrorhizon by in situ extraction and calcium alginate immobilization

Plant cell cultures of Lithospermum erythrorhizon were carried out to produce shikonin by in situ extraction and cell immobilization in calcium alginate bead in shake flask cultures. In situ product extraction and cell immobilization enhanced shikonin production and facilitated product recovery. In situ extraction by n-hexadecane and cell immobilization by calcium alginate gave higher specific shikonin productivities of 7.4 and 2.5 times, respectively, than those from the cultures of free cells without extraction. Simultaneous use of both techniques increased specific and volumetric productivities of shikonin 25- and 15-fold, respectively. In calcium alginate immobilized cell cultures, n-hexadecane addition at an early stage (before 15 days) was effective for shikonin production, and solvent addition after 15 days of the culture significantly reduced shikonin production. Higher numbers of plant cell immobilized bead inoculation did not increase shikonin production and sucrose consumption. Most of the produced shikonin was dissolved in the solvent layer.     

Caffeic acid oligomers in Lithospermum erythrorhizon cell suspension cultures

Lithospermum erythrorhizon cells cultured in pigment production (M-9) medium produced lithospermic acid B, a dimerized caffeic acid ester derivative, in quantities similar to the production of shikonin. The cells also produced a related dimer, (+)-rabdosiin. In Linsmaier-Skoog liquid medium, which suppresses shikonin production, both lithospermic acid B and (+)-rabdosiin were still formed. Lithospermic acid, a caffeic acid-rosmarinic acid conjugate, was isolated as a main constituent in Lithospermum hairy root cultures. In the aerial parts of L. erythrorhizon, the content of these phenylpropanoid oligomers was relatively low compared to that of rosmarinic acid.     

Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene

The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants. The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter. This construct was introduced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformation.The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experiment with [1,7-13C2]shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca. 20% of the overall 4HB biosynthesis in the transgenic cultures. ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.     

Effect of growth hormone modifications on shikonin production fromLithospermum erythrorhizon cell cultures within situ extraction

Summary The effect of growth hormone modifications on shikonin production was studied with the cell cultures ofLithospermum erythrorhizon. The cells grown in SH–H or SHA medium were effective for shikonin production in M–9 medium and maximum shikonin concentrations reached 43 and 63 mg/L, respectively, within situ extraction. In the case of the cells grown in SHA medium, induction time required for shikonin production was very short and the maximum shikonin concentration was obtained within 6 days.