米根霉诱导因子对紫草细胞培养中紫草宁色素分泌的影响

在紫草细胞培养中,加入米根霉粗提物可显著提高紫草宁色素产量,并可加快胞内色素分泌到培养液中的速率和数量。在细胞培养的第６天加入米根霉诱导因子时其促进紫草宁色素分泌的作用最大,培养液中紫草宁色素含量对照的２．２４倍。此外,同时加入正十六烷和米根霉诱导因子对紫草宁色素的分泌具有协同作用。     

米根霉诱导因子对紫草细胞培养中紫草宁色素分泌的影响

在紫草细胞培养中,加入采根霉粗提物可显著提高紫草宁色素产量,并可加快胞内色素分泌到培养液中的速率和数量。在细胞培养的第6天加入米根霉诱导因子时,其促进紫草宁色素分泌的作用最大,培养液中紫草宁色素含量是对照的2．24倍。此外,同时加入正十六烷和米根霉诱导因子对紫草宁色素的分泌具有协同作用。     

滇紫草愈伤组织中的紫草色素

滇紫草(Onosma paniculatum Bur.et Franch)的幼嫩根茎经二步法诱导产生的愈伤组织.含有较原植物较高的紫草色素。经薄层层析鉴定,此种紫草色素由6种单体组成,其 Rf 值与原植物中的紫草色素各类衍生物非常近似。进一步采用硅胶 H 柱层析进行分离,最后得到4种单体。经结构分析证明它们是:去氧紫色素(deoxyshikonin)、β,β-二甲基丙烯酰阿卡宁(β,β-dimethylacrylalkannin)、乙酰阿卡宁(acetylakannin)和β-乙酰氧基异戊酰阿卡宁(β-acetoxyisovalerylalkannin)。     

新疆紫草组织培养的研究进展

新疆紫草作为一种多用途的植物,它的提取物紫草宁衍生物,广泛应用于医疗、食品等方面。本文介绍了近年来国内外学者为解决新疆紫草资源短缺和保护环境所作的努力,详细介绍了新疆紫草组织培养方面的研究进展。主要包括愈伤组织的诱导及培养、细胞悬浮培养、反应器发酵培养等几方面的研究成果。     

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。     

pH值、激素对新疆紫草悬浮培养细胞生长及紫草宁衍生物合成的影响

报道了不同ｐＨ值、激素对新疆紫草悬浮培养细胞生长及紫草宁衍生物合成的影响。结果表明,新疆紫草细胞具有自我调节其培养液ｐＨ值的功能。适合于细胞生长及紫草宁衍生物形成的ｐＨ值为５．６±０．４０。ＢＡＰ、２,４－Ｄ、ＮＡＡ或ＩＢＡ对细胞生长无显著的促进作用,且都会抑制紫草宁衍生物的形成。在生长培养基中添加１．０ｍｇ／ｌ　ＩＡＡ和０．５ｍｇ／ｌＫＴ可促进细胞生长,而在生产培养基中附加０．１ｍｇ／ｌＫＴ和０．７５─１．０ｍｇ／ｌＩＡＡ则有利于紫草宁衍生物含量及产量的提高。     

不同营养因子对新疆紫草悬浮培养细胞生长及紫草宁衍生物形成的影响

报道了不同碳源、维生素、氨基酸、钙盐及肌醇对新疆紫草悬浮培养细胞生长及紫草宁衍生物形成的影响。蔗糖是最适碳源、最佳浓度为３％。Ｂ族维生素对细胞生长及紫草宁衍生物形成的促进效果不大。酪氨酸以及甘氨酸会抑制产物的形成;而１０－５ｍｏｌ／ｌ的Ｌ－苯丙氨酸以及１０－７—１０－６ｍｏｌ／ｌ维生素Ｃ可明显提高紫草宁衍生物的含量及产量。肌醇对细胞生长的影响不大,但２００ｍｇ／ｌ肌醇可促进产物的形成。适于细胞生长及紫草宁衍生物形成的钙源分别为３３２ｍｇ／ｌＣａＣｌ２·２Ｈ２Ｏ和１４００ｍｇ／ｌＣａ（Ｎｏ３）２·４Ｈ２Ｏ。文末列出了改良的生长培养基及其配方。     

用吸附法固定化培养紫草细胞

采用生物活性载体 ,通过吸附固定化方式 ,结合液体石蜡原位萃取技术 ,培养紫草细胞。测定了细胞生长、底物消耗和产物合成的动力学 ,紫草宁产率为 0 .916 g/g干重细胞和 0 .95 3g/g干重接种细胞 ,分别为悬浮培养的 12 .7倍和 6 .3倍。同时 ,对吸附与包埋固定化方法进行了综合比较 ,探讨了吸附固定化方法的应用前景。     

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。     

软紫草和黄花软紫草的核型研究

紫草科软紫草属(Arnebia Forsk.)植物约有22种,我国约产6种,其中软紫草(A. euchroma Johnst.),又名新疆紫草,是中药软紫草的主要原植物,黄花软紫草(A. guttata. Bunge),又名内蒙紫草,在内蒙古地区亦作紫草入药。在分类学上,软紫草属与紫草属(Lithospermum L.)“属间的界限不十分清楚,各家的认识不一致,种的划分也存在一些问题”,朱格麟对两个属的花、果特征进行了比较,认为两个属可以独立存在。据报道,紫草属植物的染     

Nitric Oxide Regulates Shikonin Formation in Suspension-Cultured Onosma paniculatum Cells

Endogenously occurring nitric oxide (NO) is involved in theregulation of shikonin formation in Onosma paniculatum cells.NO generated after cells were inoculated into shikonin productionmedium reached the highest level after 2 d of culture, whichwas 16 times that at the beginning of the experiment, and maintaineda high level for 6 d. A nitric oxide synthase (NOS) inhibitor,N-nitro-L-arginine (L-NNA), and a nitrate reductase (NR) inhibitor,sodium azide (SoA), consistent with their inhibition of NO biosynthesis,decreased shikonin formation significantly. This reduction couldbe alleviated or even abolished by exogenous NO supplied bysodium nitroprusside (SNP), suggesting that the inhibition ofNO biosynthesis resulted in decreased shikonin formation. However,when endogenous NO biosynthesis was up-regulated by the elicitorfrom Rhizoctonia cerealis, shikonin production was enhancedfurther, showing a dependence on the elicitor-induced NO burst.Real-time PCR analysis showed that NO could significantly up-regulatethe expression of PAL, PGT and HMGR, which encode key enzymesinvolved in shikonin biosynthesis. These results demonstratedthat NO plays a critical role in shikonin formation in O. paniculatumcells.     

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.     

Effects of Physical and Chemical Factors on Callus Growth and Shikonin Derivative Formation in the Callus Cultures of Arnebia euchroma

The effects of some physical and chemical factors on callus growth and shikonin derivative formation in the callus cultures of Arnebla euchroma were discussed. According to experiments, the optimum temperature for callus growth and shikonin derivative formation was 25℃, and the favorable initial pH of media was in the range of 5.3–5.8. Authors also found that both callus growth and shikonin derivative formation were strongly inhibited by white light. Callus growth was promoted when 0.2 mg/L IAA and 0.5 mg/L KT were added to the media, but IAA and KT did not promote shikonin derivative formation. Furthermore, the content and yield of shikonin derivatives in cultures decreased in company with the increase of IAA and KT concentration in the media.     

Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1

Shikonin is a highly lipophilic naphtoquinone found in the roots of Lithospermum erythrorhizon used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.     

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     

Anticancer Agent Shikonin Is an Incompetent Inducer of Cancer Drug Resistance

Purpose

Cancer drug resistance is a major obstacle for the success of chemotherapy. Since most clinical anticancer drugs could induce drug resistance, it is desired to develop candidate drugs that are highly efficacious but incompetent to induce drug resistance. Numerous previous studies have proven that shikonin and its analogs not only are highly tumoricidal but also can bypass drug-transporter and apoptotic defect mediated drug resistance. The purpose of this study is to investigate if or not shikonin is a weak inducer of cancer drug resistance.

Experimental Design

Different cell lines (K562, MCF-7, and a MDR cell line K562/Adr), after repeatedly treated with shikonin for 18 months, were assayed for drug resistance and gene expression profiling.

Results

After 18-month treatment, cells only developed a mere 2-fold resistance to shikonin and a marginal resistance to cisplatin and paclitaxel, without cross resistance to shikonin analogs and other anticancer agents. Gene expression profiles demonstrated that cancer cells did strongly respond to shikonin treatment but failed to effectively mobilize drug resistant machineries. Shikonin-induced weak resistance was associated with the up-regulation of βII-tubulin, which physically interacted with shikonin.

Conclusion

Taken together, apart from potent anticancer activity, shikonin and its analogs are weak inducers of cancer drug resistance and can circumvent cancer drug resistance. These merits make shikonin and its analogs potential candidates for cancer therapy with advantages of avoiding induction of drug resistance and bypassing existing drug resistance.     

Effect of Brassinolide on Growth and Shikonin Formation in Cultured Onosma paniculatum Cells

The effect of brassinolide (BR) on cell growth and shikonin and its derivative formation in Onosma paniculatum cell culture was studied. BR addition with IAA and BAP (+BR/+IAA/+BAP) in B₅ medium slightly increased the cell growth at 0.01–0.1 ppb concentration compared with a growth control (−BR/+IAA/+BAP). Only BR addition (+BR/−IAA/−BAP) at 0.001–100 ppb in B₅ medium significantly increased the cell fresh weight compared with a growth control (−BR/−IAA/−BAP). The same concentration of BR tested at 0–1000 ppb increased the cell fresh weight of +IAA/+BAP significantly more than that of −IAA/−BAP. BR at 0.001–0.1 ppb with IAA and BAP added (+BR/+IAA/+BAP) in M₉ medium increased shikonin and its derivative content markedly by 31–87%, compared with its control (−BR/+IAA/+BAP). BR at 0.001–1000 ppb without IAA and BAP added to M₉ medium (+BR/−IAA/−BAP) also increased shikonin and its derivative content compared with its control (−BR/−IAA/−BAP). However, the amount of shikonin and derivative formed of +IAA/+BAP was greater than that of −IAA/−BAP only at the same concentration of BR at 0–1 ppb. These combined results show that BR at 0.01 ppb with IAA and BAP added was the best for cell growth and shikonin formation. Formation of shikonin and its derivative by adding BR at 0.01 ppb with IAA and BAP (+BR/+IAA/+BAP) in M₉ medium was significantly enhanced 4 days after BR addition compared with a production control (−BR/+IAA/+BAP). In contrast, +BR/−IAA/−BAP vs. −BR/−IAA/−BAP was not as effective as +BR/+IAA/+BAP vs. −BR/+IAA/+BAP for the shikonin formation. The time course study for shikonin formation also showed that +BR/+IAA/+BAP and −BP/+IAA/+BAP only slightly increased cell growth in M₉ medium. Similarly, soluble protein content in the cells treated by BR at 0.01 ppb with IAA and BAP (+BR/+IAA/+BAP) exceeded that of the control (−BR/+IAA/+BAP) 4 days after BR addition. And +BR/−IAA/−BAP only slightly increased the soluble protein content over that of −BR/−IAA/−BAP. Received November 2, 1998; accepted August 25, 1999     

Shikonin inhibits the proliferation and induces the apoptosis of human HepG2 cells

This study investigated the potential of shikonin as an anticancer agent against liver cancer and an in vitro human hepatoma cancer model system. The HepG2 cell line was the hepatoma cancer model in the present study. The inhibitory effect of shikonin on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of shikonin, the cell cycle distribution, DNA fragmentation, mitochondrial membrane potential (ΔΨm) disruption, and expression of Bax and Bcl-2 were measured in HepG2 cells. The activity of shikonin in inducing apoptosis was investigated through the detection of Annexin V signal and CD95 expression by flow cytometry and electron microscopy, respectively. Shikonin inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 4.30 mg/mL. Shikonin inhibited cell growth in a dose-dependent manner and blocked HepG2 cell cycle progression at the S phase. The changes in mitochondrial morphology, dose-dependently decreased in ΔΨm, were observed in different concentrations of the drug treatment group. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression. Furthermore, we show that shikonin increases Annexin V signal and CD95 (Fas/APO) expression, resulting in apoptotic cell death of HepG2 cells. In addition, lump formation of intranuclear chromatin, pyknosis of cell nucleus, deletion of microvillus, vacuolar degeneration of mitochondria, reduction of rough endoplasmic reticulum, and resolution of free ribosome, etc., associated with apoptosis were discovered by electron microscopy in HepG2 cells after 48 h treatment. Shikonin inhibited HepG2 cells, possibly through the pathway of inducing early apoptosis, and was beneficial for restoring the apoptotic sensitivity of HepG2 cells by CD95, and should therefore be considered as a candidate agent for the prevention or treatment of human hepatoma.