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。     

Pigment formation in callus cultures of Lithospermum erythrorhizon

Undifferentiated callus tissues of Lithospermum erythrorhizon are capable of synthesizing shikonin derivatives, which are normally formed in the cork cells of the roots. Their biosynthesis in cultured cells is controlled by auxin and light. The pigment content increased linearly with time after a lag phase when callus tissues were grown on culture medium containing IAA in the dark, whereas it markedly decreased when 2,4-D was substituted for IAA or when cultures were irradiated with blue light.     

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.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

Differences in the production of shikonin derivatives by callus and suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. were examined. When Linsmaier and Skoog medium was used in suspension cultures, cell growth was not accompanied by the production of shikonin compounds. Shikonin derivatives were produced, however, when this medium was used in callus cultures. Differences in shikonin production were examined in terms of the nutrient supply, the effect of the agar itself, and the oxygen supply. Shikonin derivatives could be produced without agar by keeping the cells exposed to air while providing an adequate supply of nutrients. In callus cultures, the production of shikonin compounds was reduced remarkedly when the oxygen concentration in the atmosphere was lowered, evidence that shikonin production during L. erythrorhizon cell growth on Linsmaier and Skoog agar medium is enhanced by an abundant supply of oxygen.     

Variation in pigment production in Lithospermum erythrorhizon callus cultures

Different strains of callus cultures of Lithospermum erythrorhizon showed wide variations in the production of shikonin derivatives. From these cultures, two high pigment-producing strains, whose content of shikonin derivatives are stable and similar to that of intact plant root, have been established by repeated selection.     

Effect of nutritional factors on shikonin derivative formation in Lithospermum callus cultures

Some nutritional factors affecting the biosynthesis of shikonin derivatives in callus cultures of Lithospermum erythrorhizon were examined. High sucrose concentrations increased the content of shikonin derivatives, but neither glucose nor fructose was effective for shikonin derivative formation. High concentrations of nitrogen sources inhibited or retarded shikonin derivative formation and streptomycin sulphate stimulated their biosynthesis. Addition of ascorbic acid increased the content of shikonin derivatives. Among some precursors tested only l-phenylalanine had a positive effect. At high concentrations, Ca²⁺ and Fe²⁺ inhibited the biosynthesis of shikonin derivatives.     

Biosynthesis of shikonin in callus cultures of Lithospermum erythrorhizon

Administration of various supposed precursors to the callus cultures of Lithospermum erythrorhizon grown on the Linsmaier—Skoog medium supplemented with IAA and kinetin established that the constituent shikonin is formed via shikimic acid, p-hydroxybenzoic acid, m-geranyl-p-hydroxybenzoic acid and geranylhydroquinone. In a strain of callus culture lacking the capacity to synthesize shikonin and in callus cultures which have had this capacity but lost it due to cultivation on a medium supplemented with 2,4-D, substances up to m-geranyl-p-hydroxybenzoic acid in the biosynthetic sequence have been detected. Although illumination with white light also arrested shikonin production, traces of pigment were still formed presumably because light did not reach the innermost part of the callus cultures.     

胡萝卜细胞培养产胡萝卜素的研究

从鲜红胡萝卜主根部选取外植体,用MS型培养基诱导出愈伤组织,继代培养,周期22d,接着进行细胞的液体悬浮培养。液体培养20d时,测定β-胡萝卜素含量达41．7mg／L,占细胞干重的0．35％。培养细胞中含胡萝卜素是胡萝卜根部的3．38倍,色素比产率提高15．4倍。     

Induction of benzoquinone formation by activated carbon in Lithospermum erythrorhizon cell suspension cultures

Cultured cells of Lithospermum erythrorhizon which were capable of producing red naphthoquinone (shikonin) derivatives on Linsmaier-Skoog's agar medium stopped synthesizing these compounds when grown in liquid medium without agar. However, when the liquid medium was supplemented with a small amount of activated carbon, the cells produced a new orange benzoquinone derivative, echinofuran B, which may be considered an abnormal metabolite of geranylquinol, the key intermediate in the biosynthesis of shikonin. A similar effect of activated carbon was also observed with a variant cell line incapable of producing shikonin derivatives even on the agar medium. By contrast, the callus cultures grown on the agar medium as well as the dried roots of the intact plant were found to contain a small amount of echinofuran C, another new benzoquinone related to echinofuran B, in addition to shikonin derivatives.     

Physico-chemical factors influencing the shikonin derivatives production in cell suspension cultures of Arnebia euchroma (Royle) Johnston, a medicinally important plant species

Cell suspension cultures of Arnebia euchroma were raised from in vitro leaf-derived friable callus on liquid MS [Murashige and Skoog] medium supplemented with BAP (6-benzylaminopurine) (10.0 μM) and IBA (indole-3-butyric acid) (5.0 μM). A two-stage culture system was employed using growth and production medium for cell biomass and shikonin derivatives, respectively. Factors such as light, temperature, sucrose and pH (hydrogen ion concentration) were studied to observe their effect on the shikonin derivative production. Light conditions completely inhibited shikonin derivative production. Out of different temperature regimes tested, the highest yield (586.17 μg/g FW) was found at 25°C. Maximum production (656.14 μg/g FW) was observed in 6% sucrose. An alkaline pH (7.25-9.50) favoured shikonin derivative production. The results showed that physical and chemical factors greatly influence the production of shikonin derivatives in cell suspension cultures of A. euchroma. Therefore, by employing optimum culture conditions, it is possible to enhance the production of secondary compounds from the cells. The factors optimized for in vitro production of shikonin derivatives during the present study can successfully be employed for their large-scale production in bioreactors.     

Studies on the Naphthaquinone Constituents of Onosma confertum W. W. Smith and Quantitative Determination of Shikonin

Four compounds were isolated from Onosma confertum W. W. Smith. They were identified as deoxyshikonine (Ⅰ), β, β-dimethylacrylshkonin (Ⅱ), acetylshikonine (Ⅲ) and shikonin (Ⅳ) according to their physical and chemical constants from MS, IR, 1H-NMR, CD-HPLC determination. The total quantity of shikonin obtained after basic hydrolysis of its derivatives was determined.     

软紫草愈伤组织的初步培养

外植体来源不同的软紫草愈伤组织产生紫草色素的能力各异。６０Ｃｏ－ｒ射线辐照处理后的Ｈ２无性系愈伤组织生长量和色素产生均属上乘。改良ＭＳ基本培养基添加１毫克／升ＫＴ,０．５毫克／升ＩＡＡ,５％（Ｗ／Ｖ）蔗糖对愈伤组织培养较为适宜。马铃薯提取液对愈伤组织生长有明显的促进作用。     

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。     

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.     

γ—射线对滇紫草细胞产生色素的影响

应用５００～２５００伦琴（Ｒ）的６０钴γ射线照射滇紫草（ＯｎｏｓｍａｐａｎｉｃｕｌａｔｕｍＢｕｒ．ｅｔＦｒａｎｃｈ．）细胞系,确定了应用γ射线处理促进紫草素合成的最适辐照剂量为１５００Ｒ,处理后的细胞系在生产培养基上培养２１ｄ后紫草素含量（以干重计）达到９４．７９ｍｇ／ｇ,较对照提高了１４４６％,通过小团块选种法从中筛选到了色素含量（以干重计）高达１０３．４２ｍｇ／ｇ的高产细胞系Ｍｕｔ１,其营养生长与对照相比没有差别。     

Theoretical investigation of the radical scavenging activity of shikonin and acylshikonin derivatives

The radical scavenging activity of shikonin and acylshikonin derivatives was studied by using density functional theory. The hydrogen bond property of the studied structures was investigated using the atoms in molecules (AIM) theory. It turned out that the hydrogen bond is important for good radical scavenging activity. The hydrogen atom transfer for shikonin and acylshikonin derivatives is difficult to obtain because of the high bond dissociation energy (BDE). However, shikonin and acylshikonin derivatives appear to be good candidates for the one-electron-transfer. The introduction of acyl groups for shikonin decreases the ionization potential (IP) values compared with that of shikonin. The acylshikonin derivatives with 1H-pyrrole, furan, and thiophene groups are expected to be of the highest radical scavenging activity among the compounds investigated in this study. Taking this system as an example, we present an efficient method for the investigation of radical scavenging activity from theoretical point of view.     

藻类活性物质对紫草细胞生长和色素形成的影响

本文研究了螺旋藻、栅列藻和织线藻的水提物对新疆紫草和硬紫草细胞生长和色素形成的作用。结果表明不同种类的藻的提取物对不同紫草细胞作用呈现差异。在生长阶段,对新疆紫草,上述3种藻的低浓度提取物促进生长但作用不大,高浓度的螺旋藻和栅列藻提取物强烈抑制生长;对硬紫草,各种藻提取液的所有浓度处理均有促生长作用。在色素形成阶段,连续用高浓度织线藻提取物处理可以加速新疆紫草色素形成,同时提高色素含量。低浓度的织线藻提取液处理能提高硬紫草色素含量。栅列藻的水提物对两种紫草的色素形成均起抑制作用。螺旋藻水提物的适当浓度可加速两种紫草的色素形成。B_5培养基中加高浓度织线藻水提物,可抑制新疆紫草生长阶段的色素形成。     

紫草素的分离制备方法研究

采取CO2超临界萃取的方法从辽宁硬紫草中提取总紫草色素,用紫外分光光度法测定其含量。分别采用有机溶剂溶解碱液萃取法,直接碱水解法,以及Cu^2 络合法制备紫草素。通过三种紫草素制备方法的比较,确定先经Cu^2 络合纯化处理后再经水解法制备紫草素,收率较高,是实验室中简单、快速、高收率的由天然紫草分离制备紫草素的有效方法。     

Ethylene induced shikonin biosynthesis in shoot culture of Lithospermum erythrorhizon.

Lithospermum erythrorhizon shoots, cultured on phytohormone-free Murashige and Skoog solid medium, produced shikonin derivatives, whereas shoots cultured in well-ventilated petri dishes, produced small amount. Analysis by gas chromatography revealed the presence of ethylene in non-ventilated petri dishes where the shoots, producing shikonin derivatives, were cultured. Therefore, the possible involvement of ethylene in shikonin biosynthesis of shoot cultures was investigated. Treatment of ethylene or the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, resulted in increasing shikonin derivatives contents in cultured shoots. Silver ion, an ethylene-response inhibitor, or aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, decreased production of shikonin derivatives in cultured shoots. Our results indicate that ethylene is one of the regulatory elements of shikonin biosynthesis in L. erythrorhizon shoot culture.