Effect of jasmonates and exogenous polysaccharides on production of alkannin pigments in suspension cultures of Alkanna tinctoria

Summary The conditions for the efficient production of alkannin pigments by a suspension culture of Alkanna tinctoria were established. Pectin, polygalacturonic acid sodium salt and galactan increased the pigment production but not as much as agar did. A marked increase in the pigment content in cells and medium of suspension cultures after treatment with methyl jasmonate was observed. It was shown, applying a two-layer culture method, that mineral and olive oils intensified the pigment secretion from cells to the medium but did not enhance significantly their synthesis. Thin layer chromatography and high performance liquid chromatography methods showed that two main esters of alkannin are responsible for the characteristic colour of A. tinctoria suspension cultures.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole 3-acetic acid - NAA 1-naphthaleneacetic acid - MeJA methyl jasmonate - TLC thin layer chromatography - HPLC high performance liquid chromatography     

Micropropagation of <Emphasis Type="Italic">Arnebia hispidissima</Emphasis> (Lehm). DC. and production of alkannin from callus and cell suspension culture

Alkannin, a red-purple dye and bioactive compound found in the roots of Arnebia hispidissima has antibiotic and anti-inflammatory properties and is also used in cosmetic and textile industries at a large-scale. In the present communication, we demonstrate the establishment of callus and cell suspension culture of A. hispidissima with the aim of optimizing the production of alkannin. Highest alkannin content was recorded in cell suspension and callus culture established on M-9 medium. Production of alkannin was influenced by the different culture medium. Evaluation of alkannin content of roots of field-grown plants and in vitro grown cell, tissue and organ showed that alkannin production was higher in all in vitro grown culture systems (cell suspension, callus and roots) than the roots of field-grown plants. The present investigation may be applicable in designing systems for the large-scale cultivation of A. hispidissima cell suspensions for the production of alkannin.     

Pigment production from in vitro cultures of Alkanna tinctoria Tausch

Summary An in vitro culture of Alkanna tinctoria Tausch cells was set up in order to investigate the possibility of producing alkannin, a red naphthoquinone naturally present in the root bark of this plant. Furthermore, an in vitro culture of callusderived roots was established and the production of alkannin evaluated. In the different experimental conditions investigated, differences in the production of alkannin derivatives as well as in the type of pigments produced, were observed. The potential use of this technology is discussed.     

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     

Shikonin production and secretion by hairy root cultures of Lithospermum erythrorhizon

Summary Hairy root cultures of Lithospermum erythrorhizon were established by transformation of in vitro grown shoots with Agrobacterium rhizogenes 15834. Hairy roots cultured on Murashige and Skoog solid medium did not produce any red pigments. However, the hairy roots cultured in Root Culture solid or liquid media produced a large amount of red pigments, which were released to the medium. The addition of adsorbents to the culture medium stimulated shikonin production by ca. 3-fold. Using this method an air-lift fermenter system was established, equipped with a XAD-2 column, which continuously produced ca. 5 mg/day of shikonin during a period of more than 220 days.     

The Effective Production of Shikonin by Cultures with an Increased Cell Population

We have studied the efficient production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon with an increased cell population. The yield of shikonin derivatives was highest (800 mg/liter) when 2.8 g dry wt/liter of the cells was inoculated into the M-2 medium which we had developed for the production, but the excess inoculum lowered the yield.

We investigated suitable conditions for production with the increased cell population. The optimum amount of inoculum rose to 4.9 g dry wt/liter when the concentrations of all the components contained in the M-8 medium, which we developed for increasing the productivity by modification of the M-2 medium, were increased in proportion to the amount of inoculum, and consequently we could increase the yield of the shikonin derivatives from 1400 mg/liter to 1900 mg/liter. Moreover, the increased rate of oxygen supply in addition to the enrichment of the medium made it possible to produce 2300 mg/liter of the shikonin derivatives from a culture for which 5.6 g dry wt/liter of the cells was inoculated.     

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.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

An excellent new medium was developed for the production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon. We investigated the effects of all the components of White's medium on the production of these derivatives. Nitrate, phosphate, copper, sulfate and sucrose had especially marked effects. With the new, M-9, medium produced from these studies the yield of shikonin derivatives was 1400 mg/l and the yield for dried cells was about 12%, whereas it was 120 mg/l, or about 2% with White's medium.     

Regulation of shikonin production by glutamine in Lithospermum erythrorhizon cell cultures

Amino acid analysis has shown that Lithosperum erythrorhizon cell suspension cultures which are unable to produce shikonin derivatives in LS medium containing ammonium accumulate a large quantity of glutamine, as compared with shikonin-producing cells cultured in the production medium M9 containing nitrate as the sole nitrogen source. The addition of glutamine to M9 medium proved to be strongly inhibitory to shikonin production. Furthermore, culture experiments using an inhibitor of glutaminase suggested that shikonin synthesis is not inhibited by ammonium released from glutamine but by glutamine itself. These findings indicate that the repression of shikonin synthesis occurs in close association with an accumulation of glutamine in cultured cells grown in a medium containing ammonium.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

A two-layer culture method was established that uses an organic solvent to remove shikonin derivatives produced on cell surfaces during the culture of suspension cells of Lithospermum erythrorhizon. Some paraffins and a fatty acid ester made suitable solvents, whereas olefins and aromatic solvents extensively inhibited the production of shikonin derivatives. The yield of derivatives increased with an increase in the carbon chain length of the n-paraffin used as the solvent and when the oxygen supply was sufficient it reached the value found for the ordinary culture method.     

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.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. III. Comparison of shikonin derivatives of cultured cells and ko-shikon

A method for quantitative analysis of shikonin derivatives using high pressure liquid chromatography (HPLC) was established. With this method the composition of shikonin derivatives in cultured cells and roots of Lithospermum erythrorhizon (ko-shikon) was compared. The composition of shikonin derivatives produced by cell suspension cultures was similar to that of the ko-shikon, and the composition in cultured cells was found to fluctuate less than that of the ko-shikon.     

外循环气升式反应器培养新疆紫草细胞

采用两步培养法进行新疆紫草细胞悬浮培养及5L外循环气升式反应器扩大培养,探讨了培养过程中细胞生长、紫草色素合成与培养液的电导率、可溶性糖含量变化之间的关系。第一步培养时细胞生长迅速,但也有一部分色素合成,电导率及可溶性糖含量迅速下降;第二步培养初期电导率也开始下降,但当色素合成达到高峰并有一部分外泌到培养基后,电导率又开始回升。可溶性糖捎耗很快,到后期巳测不出其存在。因此通过监测培养液中电导率及可溶性糖的变化情况,可以为新疆紫草细胞大规模培养与色素合成提供有用的参数指标。     

Synthesis and evaluation of novel alkannin and shikonin oxime derivatives as potent antitumor agents

A set of forty alkannin and shikonin oxime derivatives were firstly designed and synthesized. Their cytotoxicities against three kinds of tumor cells and a normal cell line were tested and compared with alkannin and shikonin. The cell-based investigation demonstrated that some oxime derivatives were more or comparatively effective to the lead compounds, especially their selective and excellent antitumor activities towards K562 cells with no toxicity in normal cells. We may conclude that oximate modification to the mother nucleus of alkannin and shikonin is an available approach to acquire potent antitumor agents.     

Studies on Cell Suspension Culture and Fermentation Culture in Arnebia euchroma

This paper reports some characteristics of cell suspension and fermentation culture in Arnebia euchroma (Royle) Johnst. The yield of suspension culture reached 22.0g dry wt/L per month when inoculum quantity was 2.50 g dry wt/L. Time-course study showed that cell growith lagged in 0–3 days and enhanced greatly in 3–12 days, and almost ceased after 12 days of culture, pH value changed during the culture period and peaked on the 12th day after inoculation. When cells were cultured in liquid production medium, the contents of shikonin derivatives increased quickly and reached to the maximum about the 25th day. The cell yield of 9.47 and 9.34 g dry wt/L per month was obtained in fermentation culture. Timecourse of cell growth in fermentation culture was similar to that in suspension culture. The total content of shikonin derivatives in fermentation culture was 14.26% dry weight from 10 L bioreactor. The yield of shikonin derivatives was 1.93 g/L.     

Effects of Methyl Jasmonate on Shikonin and Dihydroechinofuran Production in Lithospermum Cell Cultures

Methyl jasmonate, when administered to Lithospermum erythrorhizoncell suspension cultures, was found to induce the productionof shikonin derivatives (the red naph-thoquinone pigments ofthe root) and dihydroechinofuran (an abnormal metabolite ofgeranylhydroquinone). Culture experiments showed that methyljasmonate caused a rapid increase in the activities of enzymesinvolved in the biosynthesis of shikonin such as p-hydroxybenzoategeran-yltransferase, which was followed by the rapid accumulationof dihydroechinofuran and the delayed production of shikonin.The induction patterns observed were similar to those elicitedby oligogalacturonides in Lithospermum cells, suggesting thatjasmonic acid or its derivative may act as a signaling moleculein the elicitation of shikonin biosynthesis. Interestingly,however, the copper ion, which is essential for inducing shikoninbiosynthesis by oligogalacturonides, was not required for shikonininduction by methyl jasmonate ¹Present address: Laboratory of Molecular & Cellular Biology,Department of Agricultural Chemistry, Kyoto University, Kitashirakawa,Kyoto, 606-01 Japan     

10升气升环流式生物反应器培养紫草细胞

本文采用自行设计研制的10升气升环流式生物反应器培养紫草细胞,培养周期34d．前14d为细胞生长培养,细胞生长呈正常的S型曲线,细胞增长到原细胞接人量的4倍．后20d为紫草色素生产培养,细胞增长到32倍。整个周期每升培养液可生产紫草色素0．6g,在反应器中,培养液pH值的变化与细胞生长呈正相关,与紫草色素的形成呈负相关,pH值变化规律可用于监测紫草细胞在生物反应器的生长和色素形成．     

Enhanced production of antitumour naphthoquinones in transgenic hairy root lines of <Emphasis Type="Italic">Lithospermum</Emphasis><Emphasis Type="Italic">canescens</Emphasis>

The effects of medium exchange and methyl jasmonate addition on growth and production of shikonin and its derivatives acetylshikonin and isobutyrylshikonin in hairy root cultures of Lithospermum canescens were investigated. Responses varied depending on the transgenic line and stage of growth at which these lines were subjected to treatment. Shikonin itself was not detected, irrespective of the transgenic line and culture treatment used. A eightfold increase in acetylshikonin and isobutyrylshikonin accumulation was achieved when 32-day-old transgenic roots of Lc1D line were transferred from LS to M9 medium for the subsequent 3 weeks of culture. Methyl jasmonate exerted a detrimental effect on red naphthoquinones production. The extracts obtained from roots cultivated in M9 medium for 3 weeks were subjected to a cytotoxicity assay and displayed cytotoxic activity against human promyelocytic leukemia cells (HL-60) at the dose of 4 μg ml⁻¹.     

Effect of l-phenylalanine on PAL activity and production of naphthoquinone pigments in suspension cultures of Arnebia euchroma (Royle) Johnst

The effects of l-phenylalanine (PHE) on cell growth and production of shikonin and its derivatives, acetylshikonin (ACS) and isobutyrylshikonin (IBS), in suspension cultures of Arnebia euchroma were examined. Supplementing media using PHE have been successfully utilized to enhance shikonin production in cell cultures of other species of Boraginaceae. l-Phenylalanine, the key compound in the phenylpropanoid pathway, is converted by phenylalanine ammonia lyase (PAL) to trans-cinnamic acid, which is the precursor of p-hydroxybenzoic acid (PHB). Coupling of PHB and geranyl pyrophosphate (derived from mevalonate pathway) by p-hydroxybenzoate-m-geranyltransferase leads later to biosynthesis of shikonins. The addition of 0.01 or 0.1?mM PHE to the culture medium stimulated cell proliferation, where the highest observed increase in fresh cell biomass (measured as a ratio of final weight to initial weight) was 12-fold, in contrast to an eightfold increase in control cultures. Whereas, growth media supplemented with 1?mM PHE markedly reduced the rate of cell growth (to only twofold). Precursor feeding had detrimental effects on both ACS and IBS production in all PHE-supplemented media. The highest total content (intracellular + extracellular) of the investigated red pigments (9.5?mg per flask) was detected in the control culture without PHE. ACS was the major component of the naphthoquinone fraction determined in cells and post-culture media. Shikonin itself was found only in the post-culture media from cultures supplemented with 0.01 or 0.1?mM PHE. Increases in PAL activity corresponded well with the accumulation of investigated naphthoquinones in control culture. However, peak PAL activity did not directly correlate with maximum production of shikonin derivatives. Cytotoxicity of extracts, prepared from the cells cultivated in the presence of PHE or in control cultures, was tested on three cancer cell lines: HL-60, HeLa, and MCF-7. The extracts prepared from the untreated control cultures proved to be the most potent against the examined cancer cell lines. The mean inhibitory concentration values were 0.3, 13, and 8???g?ml^?1 for the HL-60, HeLa, and MCF-7 cells, respectively.