Shikonin Production Research Articles

Pharmacological Significance of Boraginaceae with Special Insights into Shikonin and Its Potential in the Food Industry.

Shikonin is a naphthoquinone pigment present in the hairy roots of the plant species from the Boraginaceae family. The compound has been well investigated for its highly efficient medicinal, antioxidant, and antimicrobial properties. Various extraction methodologies have been employed to maximise yield while minimising waste production of shikonin and its derivatives. Despite substantial research on shikonin and Boraginaceae plants, a research gap persists in the food industry and extraction technologies. This review addresses crucial aspects of shikonin deserving of further exploration. It begins by elucidating the attributes of the Boraginaceae plants and their medicinal traits in folklore. It proceeds to focus on the roots of the plant and its medicinal properties, followed by extraction procedures explored in the last fifteen years, emphasising the novel technologies that have been chosen to improve the yield extract while minimising extraction times. Furthermore, this review briefly outlines studies employing cell culture techniques to enhance in vitro shikonin production. Lastly, attention is directed towards research in the food industry, particularly on shikonin-loaded biodegradable films and the antioxidant activity of shikonin. This review concludes by summarising the future potential in food science and prominent research gaps in this field.

Peroxisomal 4-coumaroyl-CoA ligases participate in shikonin production in Lithospermum erythrorhizon.

4-Coumaroyl-CoA ligase (4CL) is a key enzyme in the phenylpropanoid pathway, which is involved in the biosynthesis of various specialized metabolites such as flavonoids, coumarins, lignans, and lignin. Plants have several 4CLs showing divergence in sequence: Class I 4CLs involved in lignin metabolism, Class II 4CLs associated with flavonoid metabolism, and atypical 4CLs and 4CL-like proteins of unknown function. Shikonin, a Boraginaceae-specific specialized metabolite in red gromwell (Lithospermum erythrorhizon), is biosynthesized from p-hydroxybenzoic acid, and the involvement of 4CL in its biosynthesis has long been debated. In this study, we demonstrated the requirement of 4CL for shikonin biosynthesis using a 4CL-specific inhibitor. In silico analysis of the L. erythrorhizon genome revealed the presence of at least 8 4CL genes, among which the expression of 3 (Le4CL3, Le4CL4, and Le4CL5) showed a positive association with shikonin production. Phylogenetic analysis indicated that Le4CL5 belongs to Class I 4CLs, while Le4CL3 and Le4CL4 belong to clades that are distant from Class I and Class II. Interestingly, both Le4CL3 and Le4CL4 have peroxisome targeting signal 1 in their C-terminal region, and subcellular localization analysis revealed that both localize to the peroxisome. We targeted each of the 3 Le4CL genes by CRISPR/Cas9-mediated mutagenesis and observed remarkably lower shikonin production in Le4CL3-ge and Le4CL4-ge genome-edited lines compared with the vector control. We, therefore, conclude that peroxisomal Le4CL3 and Le4CL4 are responsible for shikonin production and propose a model for metabolite-specific 4CL distribution in L. erythrorhizon.

The Ammonium/Nitrate Ratio Affects the Growth and Shikonin Accumulation in Arnebia euchroma

Nitrogen (N) strongly affects plant growth and metabolism. Although ammonium toxicity has been reported, the effects of nitrogen on shikonin biosynthesis remain obscure. In this study, we tested four different concentrations of NH4+ on Arnebia euchroma hairy roots (AEHR) to clarify the influence of NH4+ on the growth of AEHR and on shikonin accumulation in them and the possible mechanisms. The results showed that compared with the 0% NH4+ treatment (only nitrate as a nitrogen source), the 10% NH4+ treatment increased the fresh weight and the dry weight of AEHR and promoted the synthesis of shikonins. In contrast, the 20% NH4+ treatment started to show inhibition effects on the growth of and shikonin accumulation in AEHR, and the 30% NH4+ treatment exhibited the strongest inhibition effects. With an increased percentage of NH4+, the AEHR became shorter and thicker, with more branches. To further elucidate the mechanisms, we analyzed the time course of nitrogen assimilation, the gene expression level of key enzymes involved in shikonin biosynthesis pathway, and the content of various endogenous hormones in the presence of toxic NH4+ concentrations. The results indicated that auxin and cytokinin might regulate the growth and architecture of AEHR under NH4+ toxicity and revealed that the jasmonate level was reduced along with the inhibition of shikonin biosynthesis. This first comprehensive investigation of the effects of the ammonium/nitrate ratio on shikonin biosynthesis not only provides valuable data for optimizing the in vitro culture of A. euchroma and its shikonin production, but also suggests potential fertilizing strategies for its cultivation.

Temporary immersion bioreactors as a useful tool for obtaining high productivity of phenolic compounds with strong antioxidant properties from Pontechium maculatum

Pontechium maculatum (Russian bugloss) is a medical plant belonging to the family Boraginaceae. Although this species is known as a medical plant rich in biologically active secondary metabolites, biotechnological studies about this valuable plant is still missing. The scientific objectives of this study were to investigate the biomass production, synthesis, and productivity of various phenolic acids, flavonoids, and shikonin in P. maculatum cultivated in various breeding systems. Additionally, the antioxidant activity of plant-derived extracts was evaluated. Plants were cultivated in a traditional agar-solidified medium, a liquid medium with rotary shaking, and a temporary immersion bioreactors Plantform™ (TIB), as well as cultivated in soil (ex vitro conditions). Analyses of the growth index and dry weight accumulation were performed on the collected material. In the extracts obtained from examined plants, total phenolic content was estimated, and qualitative and quantitative analysis of phenolic derivatives using DAD-HPLC was conducted, simultaneously with an analysis of antioxidant capacity. TIB stimulated the highest synthesis of all examined phenolic acids and shikonin. In TIB-cultivated shoots level of rosmarinic acid obtained a concentration of 3160.76 mg × 100 g− 1 dry weight (DW), and shikonin obtained a concentration of 77.26 mg × 100 g− 1 DW. Furthermore, plants from TIB were characterized by the highest productivity of all studied phenolic derivatives, what makes it very effective platform for the synthesis of biologically active secondary metabolites in Russian bugloss. Moreover, this article shows that P. maculatum is a rich source of various phenolic derivatives with high antioxidant potential.

Characterization of Arnebia euchroma PGT homologs involved in the biosynthesis of shikonin

Shikonin is a red naphthoquinone natural product from plants with high economical and medical values. The para-hydroxybenzoic acid geranyltransferase (PGT) catalyzes the key regulatory step of shikonin biosynthesis. PGTs from Lithospermum erythrorhizon have been well-characterized and used in industrial shikonin production. However, its perennial medicinal plant Arnebia euchroma accumulates much more pigment and the underlying mechanism remains obscure. Here, we discovered and characterized the different isoforms of AePGTs. Phylogenetic study and structure modeling suggested that the N-terminal of AePGT6 contributed to its highest activity among 7 AePGTs. Indeed, AePGT2 and AePGT3 fused with 60 amino acids from the N-terminal of AePGT6 showed even higher activity than AePGT6, while native AePGT2 and AePGT3 don't have catalytic activity. Our result not only provided a mechanistic explanation of high shikonin contents in Arnebia euchroma but also engineered a best-performing PGT to achieve the highest-to-date production of 3-geranyl-4-hydroxybenzoate acid, an intermedium of shikonin.

A simple protocol for high frequency plant regeneration and enhancing Shikonin production from callus cultures in Arnebia hispidissima

A rapid and efficient protocol for callus induction, enhancing Shikonin pigment production and plant regeneration from various explants such as leaf, shoot tip, internodal and nodal explants is presented. Highest callus induction efficiency of 86.7% per explant was obtained with nodal explants on MS medium blended with 0.5 mgl−1 KIN, 1.0 mgl−1 2, 4-D and 100 mgl−1CH medium. Callus from nodal explants grew faster and showed improved potentiality for shoot regeneration (80%) on MS medium supplemented with 0.5 mgl−1 KIN, 0.25 mgl−1 BAP, 0.1 mgl−1 IAA and 100 mgl−1 CH. MS medium added with 2.0 mgl−1 IBA efficiently induced roots in proliferating shoots further those were transferred to soil. Greatest response obtained for callus induction for Shikonin production was 80.0% from nodal segments, 46.7% from shoot tip explants followed by an intermediate response of 66.7% from internodal and leaf segment cultured on media combination MS + mgl−1 BAP 0.25 + 1.0 mgl−1 IAA + 100 mgl−1 CH. Shikonin pigment production was stimulated upon transfer of callus tissues to shoot induction medium containing IAA, BAP and KIN instead of 2, 4-D under 12 h dark period. MS medium supplemented with 0.25 mgl−1 BAP, 1.0 mgl−1 IAA and 100 mgl−1 CH was found to be ideal for high Shikonin induction in callus cultures of all the explants employed. UV–visible spectrophotometry quantified Shikonin content of 0.50 mgg−1 in fresh callus after 50 days of culture. Thus, a reproducible protocol for establishment of fast growing callus with high frequency plant regeneration from nodal segments of Arnebia hispidissima which could serve as a reliable source for enhanced production of Shikonin for pharmaceuticals, medicinal and food industry has been presented.

In Vitro CALLUS PROLIFERATION FOR PRODUCTION OF SHIKONIN DERIVATIVES AND PROTEIN PROFILING IN Arnebia hispidissima (Lehm.) A.DC.

Arnebia hispidissima (Lehm.) A.DC. (Boraginaceae), a red dye-yielding and medicinally important plant, is commonly distributed in arid/semi-arid regions of Rajasthan. This paper highlights an in vitro callus proliferation protocol for production of shikonin and its derivatives. Immature seeds explants were found to be most favorable for callus induction (74.6±1.52%). During callus induction, the maximum percentage response (78.9±1.57) was achieved on Murashige and Skoog (MS, 1962) medium with additives supplemented with NAA (1.0 mg L-1) and 2, 4-D (2.0 mg L-1). Dye producing red colored globular and fast-growing (four-fold) callus was achieved on 2, 4-D (0.1 mg L-1), Kin (0.5 mg L-1) and BAP (0.1 mg L-1). In vivo root extract and in vitro proliferated callus extract were compared using thin layer chromatography (TLC). Six and four promising bands were recorded from in vivo root extract and in vitro callus extract, respectively on the Methanol : Chloroform (1:9) extraction system. Three different types of calli (red, dark red and green) growing on varying media composition were studied for differential gene expression using SDS – PAGE which revealed presence of seven, twelve and twelve protein bands, respectively. The presence of one extra band (21.4 kDa) in dark red callus might be responsible for dye/shikonin derivative production. The results obtained in this study are preliminary and requires further analysis of protein profile/proteome in order to understand the shikonin production mechanism in this plant.

Integrative analysis of the shikonin metabolic network identifies new gene connections and reveals evolutionary insight into shikonin biosynthesis.

SummaryPlant specialized 1,4-naphthoquinones present a remarkable case of convergent evolution. Species across multiple discrete orders of vascular plants produce diverse 1,4-naphthoquinones via one of several pathways using different metabolic precursors. Evolution of these pathways was preceded by events of metabolic innovation and many appear to share connections with biosynthesis of photosynthetic or respiratory quinones. Here, we sought to shed light on the metabolic connections linking shikonin biosynthesis with its precursor pathways and on the origins of shikonin metabolic genes. Downregulation of Lithospermum erythrorhizon geranyl diphosphate synthase (LeGPPS), recently shown to have been recruited from a cytoplasmic farnesyl diphosphate synthase (FPPS), resulted in reduced shikonin production and a decrease in expression of mevalonic acid and phenylpropanoid pathway genes. Next, we used LeGPPS and other known shikonin pathway genes to build a coexpression network model for identifying new gene connections to shikonin metabolism. Integrative in silico analyses of network genes revealed candidates for biochemical steps in the shikonin pathway arising from Boraginales-specific gene family expansion. Multiple genes in the shikonin coexpression network were also discovered to have originated from duplication of ubiquinone pathway genes. Taken together, our study provides evidence for transcriptional crosstalk between shikonin biosynthesis and its precursor pathways, identifies several shikonin pathway gene candidates and their evolutionary histories, and establishes additional evolutionary links between shikonin and ubiquinone metabolism. Moreover, we demonstrate that global coexpression analysis using limited transcriptomic data obtained from targeted experiments is effective for identifying gene connections within a defined metabolic network.

Cloning and functional analysis of EpGHQH1 in shikonin production of Echium plantagineum

Cloning and functional analysis of EpGHQH1 in shikonin production of Echium plantagineum

Establishment of the hairy root culture of Echium plantagineum L. and its shikonin production.

Echium plantagineum L. (Boraginaceae) is an invasive species in Australia and contains medicinal shikonins in its roots. In this study, the hairy root lines of E. plantagineum were established using Agrobacterium rhizogenes strain ATCC15834 and confirmed by the amplification of the rolB gene. Results showed significant difference in shikonin production between the hairy root lines in the 1/2B5 and M9 media. The biomass of the lines in the 1/2B5 medium was fivefold of that in the M9 medium. However, the components of detected shikonins were similar in these two liquid media. By contrast, different accumulation profiles appeared in the hairy root lines. HPLC analysis revealed the presence of nine possible related compounds, including shikonins, and acetylshikonin was the most abundant shikonin derivative. The content of acetylshikonin in the 1/2B5 medium (36.25mg/L on average) was twofold of that in the M9 medium. Our results showed that the hairy root cultures of E. plantagineum can be used in enhancing the production of potential pharmaceutical compounds, such as acetylshikonin.

Apple latent spherical virus (ALSV)-induced gene silencing in a medicinal plant, Lithospermum erythrorhizon

Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have been shown advantageous in introducing iRNA genes into non-model plants. This study describes the development of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRNA was detected in infected plants but not in escape and mock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the VIGS system silence target genes in soil-grown L. erythrorhizon.

Establishment of RNA interfered hairy root system of two CYP450 genes in Arnebia euchroma and its influence

In this study, based on the transcriptome database of suspension cells of Arnebia euchroma, we explored two candidate cytochrome P450 enzyme genes that might relate to the shikonin biosynthesis downstream pathway when CYP76B74 sequence was referenced. We constructed interference-type hairy roots of candidate genes and cultured them. We measured the fresh weight, dry weight, total naphthoquinone content, shikonin and its derivatives content and expression levels of key enzyme genes involved in shikonin biosynthesis pathway. The effects of candidate genes on the growth and shikonin production of A. euchroma hairy roots were discussed, and the possible regulatory mechanisms that candidate genes affected shikonin synthesis were discussed. Through local Blast and phylogenetic analysis, two candidate CYP450 genes(CYP76B75 and CYP76B100) with high homology to CYP76B74 in A. euchroma were screened, and corresponding interference hairy roots were constructed. Compared with the control(RNAi-control), the fresh weight of CYP76B75 interfered hairy root(RNAi-CYP76B75) and CYP76B100 interfered hairy root(RNAi-CYP76B100) were significantly reduced, while dry weight were not affected, so the dry rate increased significantly. Except for β-acetoxyisovalerylalkannin, which is high in three groups of hairy roots, the contents of shikonin, deoxyshikonin, acetylshikonin, β,β'-dimethacrylicalkannin, β-hydroxyisovalerylshikonin,β-hydroxyisovalerylshikonin, isobutyrylshikonin and total naphthoquinones showed a consistent pattern: RNAi-CYP76B75>RNAi-CYP76B100>RNAi-control. Among them, the synthesis of β-hydroxyisovalerylshikonin was most significantly promoted by interfering with the expression of CYP76B75. The content of β-hydroxyisovalerylshikonin in RNAi-CYP76B75 was 11.7 times that of RNAi-control. RESULTS:: of real-time qPCR analysis showed that compared to RNAi-control, the expression levels of AePGT gene in RNAi-CYP76B75 and RNAi-CYP76B100 were not changed significantly, and the expression levels of CYP76B74 and AeHMGR were up-regulated. In addition, the expression level of CYP76B100 in RNAi-CYP76B75 was down-regulated, whereas in RNAi-CYP76B100, the expression of CYP76B75 was significantly up-regulated. Therefore, this study confirmed that when the expression of CYP76B75 and CYP76B100 were interrupted, the growth of hairy roots were suppressed, but the synthesis of shikonin were promoted. They might increase the shikonin biosynthesis by up-regulating the expression of CYP76B74 in the hairy roots of A. euchroma.

Hybrid de novo genome assembly of red gromwell (Lithospermum erythrorhizon) reveals evolutionary insight into shikonin biosynthesis

Lithospermum erythrorhizon (red gromwell; zicao) is a medicinal and economically valuable plant belonging to the Boraginaceae family. Roots from L. erythrorhizon have been used for centuries based on the antiviral and wound-healing properties produced from the bioactive compound shikonin and its derivatives. More recently, shikonin, its enantiomer alkannin, and several other shikonin/alkannin derivatives have collectively emerged as valuable natural colorants and as novel drug scaffolds. Despite several transcriptomes and proteomes having been generated from L. erythrorhizon, a reference genome is still unavailable. This has limited investigations into elucidating the shikonin/alkannin pathway and understanding its evolutionary and ecological significance. In this study, we obtained a de novo genome assembly for L. erythrorhizon using a combination of Oxford Nanopore long-read and Illumina short-read sequencing technologies. The resulting genome is ∼367.41 Mb long, with a contig N50 size of 314.31 kb and 27,720 predicted protein-coding genes. Using the L. erythrorhizon genome, we identified several additional p-hydroxybenzoate:geranyltransferase (PGT) homologs and provide insight into their evolutionary history. Phylogenetic analysis of prenyltransferases suggests that PGTs originated in a common ancestor of modern shikonin/alkannin-producing Boraginaceous species, likely from a retrotransposition-derived duplication event of an ancestral prenyltransferase gene. Furthermore, knocking down expression of LePGT1 in L. erythrorhizon hairy root lines revealed that LePGT1 is predominantly responsible for shikonin production early in culture establishment. Taken together, the reference genome reported in this study and the provided analysis on the evolutionary origin of shikonin/alkannin biosynthesis will guide elucidation of the remainder of the pathway.

Molecular identification and phytochemical screening of endophytic fungi isolated from Lithospermum officinale L. roots: A new source of shikonin

Endophytic fungi are microorganisms located in the inter- or intracellular compartments of plant tissues but with no harmful effects. They are considered a potential source of biological compounds. The present study was conducted to investigate the molecular identification of endophytic fungi isolated from the roots of Lithospermum officinale and their potential production of shikonin. Phylogenetic analysis was performed based on the Internal Transcribed Spacer (ITS) region and the isolates were classified into five genera as follows: Alternaria, Chaetosphaeronema, Fusarium, Mucor, and Trichoderma. The study on the methanol extracts of endophytic fungi indicated that total polyphenol content had a positive relationship with antioxidant activities and the highest antioxidant activity belonged to the methanol extracts of Fusarium tricinctum and Alternaria altenata. Then, to investigate the ability of the fungal isolates to produce shikonin, a naphthoquinone compound with high biological activity, the extracts were subjected to HPLC. The results obtained from HPLC-mass spectrometry showed that shikonin could be produced only by F. tricinctum. Thus, F. tricinctum isolated from the roots of L. officinale can be presented as a new source of shikonin.

Shikonin Production by Callus Culture of Onosma bulbotrichom as Active Pharmaceutical Ingredient.

The objective of this research was in-vitro germination and callus induction of Onosma bulbotrichum (O.bulbotrichum) as a medicinal herb which belongs to Boraginaceae family. For germination, the seeds were cultured on growth regulator-free MS medium and for callus induction, seeds were sown on modified MS medium containing different concentrations of kinetin (kn)- Indole-3-acetic acid (IAA) and kn- 2,4-D (2,4-dichlorophenoxyacetic acid), respectively. The plates were maintained in the dark at growth chamber. After 7 days seed germination on hormone-free medium and after 10 days callus initiation on modified medium in the presence of hormones was occurred. The maximum pigmented callus (100%) was observed on modified MS medium with a combination of 0.2 mg.L-1 IAA + 2.10 mg.L-1 kn. Shikonin determination was performed by HPLC method. In addition, total hydroxynaphtoquinons as polyphenols in sum of callus and culture medium were measured by spectrophotometric method and revealed that total naphtoquinones content at IAA was more than 2, 4-D.

Potential and Prospects of Shikonin Production Enhancement in Medicinal Plants

Plants are a rich source of valuable chemicals that are used as herbal and modern medicines. Shikonin, a naphthoquinone red pigment, produced from roots of Arnebia hispidissima, A. euchroma, Lithospermum erythrorhizon and other Boraginaceous species was the first phytochemical to be produced on commercial scale using biotechnological approach. Plant cell culture technologies were the only tools employed for secondary metabolites production in other cases in past. Shikonin production and localization is tissue specific with stringent metabolic regulation. Hence, the differentiated cultures such as those of hairy root cultures offer a great promise for secondary metabolite production. A. rhizogenes-mediated transformed roots are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce larger amounts of secondary metabolites. The present review deliberates upon potential and prospects of diverse physico-chemical factors that affect shikonin production in tissue culture, as well as, the genetic modification for enhancement of shikonin production.

Involvement of LeMDR, an ATP-binding cassette protein gene, in shikonin transport and biosynthesis in Lithospermum erythrorhizon

BackgroundShikonin is a naphthoquinone secondary metabolite with important medicinal value and is found in Lithospermum erythrorhizon. Considering the limited knowledge on the membrane transport mechanism of shikonin, this study investigated such molecular mechanism.ResultsWe successfully isolated an ATP-binding cassette protein gene, LeMDR, from L. erythrorhizon. LeMDR is predominantly expressed in L. erythrorhizon roots, where shikonin accumulated. Functional analysis of LeMDR by using the yeast cell expression system revealed that LeMDR is possibly involved in the shikonin efflux transport. The accumulation of shikonin is lower in yeast cells transformed with LeMDR-overexpressing vector than that with empty vector. The transgenic hairy roots of L. erythrorhizon overexpressing LeMDR (MDRO) significantly enhanced shikonin production, whereas the RNA interference of LeMDR (MDRi) displayed a reverse trend. Moreover, the mRNA expression level of LeMDR was up-regulated by treatment with shikonin and shikonin-positive regulators, methyl jasmonate and indole-3-acetic acid. There might be a relationship of mutual regulation between the expression level of LeMDR and shikonin biosynthesis.ConclusionsOur findings demonstrated the important role of LeMDR in transmembrane transport and biosynthesis of shikonin.

Metabolic Profiling and Identification of Shikonins in Root Periderm of Two Invasive Echium spp. Weeds in Australia.

Metabolic profiling can be successfully implemented to analyse a living system’s response to environmental conditions by providing critical information on an organism’s physiological state at a particular point in time and allowing for both quantitative and qualitative assessment of a specific subset(s) of key metabolites. Shikonins are highly reactive chemicals that affect various cell signalling pathways and possess antifungal, antibacterial and allelopathic activity. Based on previous bioassay results, bioactive shikonins, are likely to play important roles in the regulation of rhizosphere interactions with neighbouring plants, microbes and herbivores. An effective platform allowing for rapid identification and accurate profiling of numerous structurally similar, difficult-to-separate bioactive isohexenylnaphthazarins (shikonins) was developed using UHPLC Q-TOF MS. Root periderm tissues of the invasive Australian weeds Echium plantagineum and its congener E. vulgare were extracted overnight in ethanol for shikonin profiling. Shikonin production was evaluated at seedling, rosette and flowering stages. Five populations of each species were compared for qualitative and quantitative differences in shikonin formation. Each species showed little populational variation in qualitative shikonin production; however, shikonin was considerably low in one population of E. plantagineum from Western New South Wales. Seedlings of all populations produced the bioactive metabolite acetylshikonin and production was upregulated over time. Mature plants of both species produced significantly higher total levels of shikonins and isovalerylshikonin > dimethylacrylshikonin > shikonin > acetylshikonin in mature E. plantagineum. Although qualitative metabolic profiles in both Echium spp. were nearly identical, shikonin abundance in mature plant periderm was approximately 2.5 times higher in perennial E. vulgare extracts in comparison to those of the annual E. plantagineum. These findings contribute to our understanding of the biosynthesis of shikonins in roots of two related invasive plants and their expression in relation to plant phenological stage.

Lithospermum erythrorhizon cell cultures: Present and future aspects.

Lithospermum erythrorhizon cell cultures have been used to produce plant secondary metabolites, as well as in biosynthetic studies. Shikonin, a representative secondary metabolite of L. erythrorhizon, was first produced industrially by dedifferentiated cell cultures in the 1980s. This culture system has since been used in research on various plant secondary metabolites. Other boraginaceaeous plant species, including Arnebia, Echium, Onosma and Alkanna, have been shown to produce shikonin, and studies have assessed shikonin regulation, including transgene expression, in these plants. This review summarizes current knowledge of shikonin production by L. erythrorhizon cell and hairy root cultures, including the historical aspect of large-scale production, and discusses future biochemical and biological research using this species.

Characterization of Shikonin Derivative Secretion in Lithospermum erythrorhizon Hairy Roots as a Model of Lipid-Soluble Metabolite Secretion from Plants.

Shikonin derivatives are specialized lipophilic metabolites, secreted in abundant amounts from the root epidermal cells of Lithospermum erythrorhizon. Because they have anti-microbial activities, these compounds, which are derivatives of red naphthoquinone, are thought to serve as a chemical barrier for plant roots. The mechanism by which they are secreted from cells is, however, largely unknown. The shikonin production system in L. erythrorhizon is an excellent model for studying the mechanism by which lipophilic compounds are secreted from plant cells, because of the abundant amounts of these compounds produced by L. erythrorhizon, the 0 to 100% inducibility of their production, the light-specific inhibition of production, and the visibility of these products as red pigments. To date, many factors regulating shikonin biosynthesis have been identified, but no mechanism that regulates shikonin secretion without inhibiting biosynthesis has been detected. This study showed that inhibitors of membrane traffic strongly inhibit shikonin secretion without inhibiting shikonin production, suggesting that the secretion of shikonin derivatives into the apoplast utilizes pathways common to the ADP-ribosylation factor/guanine nucleotide exchange factor (ARF/GEF) system and actin filament polymerization, at least in part. These findings provide clues about the machinery involved in secreting lipid-soluble metabolites from cells.