Shikonine

Shikonin [517-89-5] (Cl 75535) occurs as an acetyl derivative in the Japanese shikone, Uthospermum eTythrorhi n another member of the Boraginaceae family. It is the (R)-optical isomer of alkannin (66). Tissue cultures of E. eythrorhi n are used in Japan to manufacture shikonin mainly for cosmetic use (67). Both alkannin and shikonin are mordant dyes producing violet to gray colors on fabrics. In Japan, shikonin was used to dye fabrics a color known as Tokyo Violet. Shikalkin [54952-43-1] the racemate (11), has been synthesized (68). 

Plant cell suspensions offer the potential to produce valuable phytochemicals, traditionally extracted from the naturally grown whole plant, under controlled and reproducible conditions. To date, commercial processes involving these systems have been limited to just a handful of applications, including the much-cited shikonin [1] and ginseng [2,3]. 

Low-energy ultrasound was employed to increase by up to 70% the production of shikonin (Fig. 4) in cell cultures of the medicinal herb Lithospermum erythrorhizon. Shikonin exhibits a variety of effects, which includes anti-inflammatory, antigonadotropic and human immunodeficiency virus type 1 (HIV-1) suppression activities. 

Lin LD, Wu JY. (2002) Enhancement of shikonin production in single- and two-phase suspension cultures of Lithospermum erythrorhizon cells using low-energy ultrasound. Biotechnol Bioeng 78 81-88. 

Chen, X, Yang, L, Zhang, N, Turpin, JA, Buckheit, RW, Osterling, C, Oppenheim, JJ, Howard, OMZ. (2003) Shikonin, a component of Chinese herbal medicine, inhibits chemokine receptor function and suppresses Human Immunodeficiency Virus type Antimicrob Agents Chemother 47 2810-2816. 

Geminal dihalides have also been applied for the construction of 1,3-dioxanes and congeners. For example, bromochloromethane readily reacted with tetrahydroxynaphthalenes to afford the tetracycle 218 in good yield (Equation 82). Bisdioxane 218 was subsequently used for the synthesis of alkannin and shikonin <1998AGE839, 2000SC1023>. 

Arnebia euchroma Forssk. Zi Cao (root) Shikonin, acetylshikonin, beta-beta-dimethylacrytoylshikonin, beta-OH-isovalerylshikonin, alkamin-B, beta-di-Me-acrylate.33-450 Anti-inflammatory, antiseptic, antibacterial, toothache, eye diseases, ahealerof cuts, bums, and wounds. 

Lithospermum erythrorhizon Sieb. et Zucc. L. officinalis var. erythrorhizon Sieb. et Zucc. Zhu Cao (Groomwell) (root) Quinonoid, alkannan, acetylshikonin, shikonin, lithospermin, dihydroshikonin, cycloshikonin.1-69 Ointment to treats wounds and bums, antitumor, antipyretic, regulating blood circulation, diuretic, purgative, remedy for smallpox. 

Alkannin. shikonin. and shikalkin are grouped together because the first two are enantiomers and the last one is their racemate. Alkannin (Cl Natural Red 20 Cl 75530) [Anchu.sa tinctoria or ttlkanua tinctoria) is a member uf the Boraginaceae family. It is found in the roots of alkanet, a perennial shrub native to Southern Eurupe. 

Others L-Dopa, ginsengoside, shikonin, rosmarinic acid, saponin, ubiqumone-10, diosgenin... 

After cells reach a certain population, the modification of the medium may affect product accumulation. Fujita et al. (1981) improved the yield of shikonin derivatives from the suspension cultures of Lithospermum erythrorhizon by using a production medium. The production medium usually contains more sucrose but less inorganic components and vitamins than the growth medium. 

Fujita, Y., Y. Kara, C. Suga, and T. Morimoto, "Production of Shikonin Derivatives by Cell Suspension Cultures of Lithospermum erythrorhizon II. A New Medium for the Production of Shikonin Derivatives," Plant Cell Reports 1(1981 ) 61-63. 

First commercial process utilizing plant cells to manufacture shikonin was developed by a Japanese firm. Mitsue Petrochemical Industries Ltd. in 1983. 

Some plant cells can be encouraged to grow in vitro under specific controlled conditions, which can be used to produce secondary plant metabolites at higher yields than those found in plants, but at a high cost. There are few examples where the technological difficulties and costs involved have led to commercialisation currently, but the valuable pharmaceutical alkaloid berberine, the quinnone shikonin and the terpenoid paclitaxel are all potential candidates for production by cell culture. 

Several SM have been used by mankind for thousands of years22,27 as dyes (e.g., indigo, shikonine), flavors (e.g., vanillin, capsaicin, mustard oils), fragrances (e.g., rose oil, lavender oil and other essential oils), stimulants (e.g., caffeine, nicotine, ephedrine), hallucinogens (e.g., morphine, cocaine, mescaline, hyoscyamine, scopolamine, tetrahydrocannabinol), insecticides (e.g., nicotine, piperine, pyrethrin), vertebrate and human poisons (e.g., coniine, strychnine, aconitine) and even therapeutic agents (e.g., atropine, quinine, cardenolides, codeine, etc.). 

Cell line selection is one of the traditional and effective approaches to enhancing metabolite accumulation, and biochemical studies provide the fundamental information for the intentional regulation of secondary metabolism in plant cells. In a carrot suspension culture regulated by 2,4-dichlorophenoxyace-tic acid, Ozeki et al. [7] found that there was a correlation between anthocyanin synthesis and morphological differentiation for somatic embryogenesis they also demonstrated the induction and repression of phenylalanine ammonia lyase (PAL) and chalcone synthase correlated with formation of the respective mRNAs. Two biosynthetic enzymes, i. e., PAL and 3-hydroxymethylglutaryl-CoA reductase, were also related with shikonin formation in Lithospermum erythro-rhizon cultures [8]. 

Although plant cell culture has been demonstrated to be a useful method for the production of valuable secondary metabolites, many problems arise during bioprocess scale-up (Table 2). At present, there are only a few industrial processes in operation using plant cell cultures, which include shikonin, ginseng... 

Nitrogen source is also very important for plant cell metabolite formation, as reported in suspension cultures of Holarrhena antidysenterica for accumulation of alkaloids [36], in cell suspensions of Vitis vinifera for anthocyanin formation [37], and in shikonin production by Lithospermum erythrorhizon cell cultures [38]. 

Some secondary metabolites have been observed in much higher concentrations in cultured cells than in whole plants of the same species. These include ginsengosides from Panax ginseng (27% of cell dry weight in culture, 4.5% in whole plants), anthraquinones from Morinda citrafolia (18% in culture, 2.2% in plants) and shikonin from Lithospernum erythrorhizon (12 % in culture, 1.5 % in plants) [19,20]. 

Specific production rate tends to be lower. For example, despite several years of optimization studies, volumetric productivity of shikonin by suspension cultures of L. erythrorhizon was reported as 0.1 gprodUct 1 1 d 1 [23]. For com-... 

Both cell culture with a lipophilic extraction phase and with a polar extraction phase have been reported to be helpful for the accumulation and detection of secondary substances [7,8]. Plant cell cultures release lipophilic and volatile substances such as ethylene, ethanol, and acetaldehyde. The addition of a lipophilic phase to the culture medium can be used as a means of accumulating and detecting these substances. Maisch et al. [8] found that the addition of XAD-4 resin to Nicotiana tabacum cultures enhanced the production of phenolic secondary metabolites several times compared to the adsorbent-free control. Kim and Chang [9] reported in situ extraction for enhanced shikonin production by Lithospermum erythrorhizon. When n-hexadecane was added to the cultivation, higher specific shikonin productivity was obtained than that from the cultures of free cells without extraction. They also suggested that n-hexadecane addition at an early stage in calcium alginate immobilized cell cultures was effective for shikonin production. Most of the produced shikonin was dissolved in n-hexadecane, so it would reduce the costs for shikonin separation. 

In spite of these problems, one product of plant cell suspension culture has achieved commercialization. This product, shikonin, is a red dye, produced in Japan at the level of several tons per year. Published reports suggest the optimum yield occurs at a relatively low volumetric mass transfer for oxygen, roughly 10 hr-1 (see Fig. 1). When higher oxygen transfer rates are used, the production rate drops. 

This suggested that the plant cells producing shikonin were shear sensitive, which we have confirmed in our laboratory. We investigated plant cell suspension culture under fixed shear conditions. When insoluble oxygen carriers were added, in our case a perfluorocarbon, significant improvements occurred in the cell and product yield. 

Figure 1. Relationship between the yield of shikonin and initial k a. Reprinted with permission from Tanaka [2],...

With shikonin, shear effects bein to limit productivity at a kLa of only 10 hr 1 in a shake flask culture. Therefore, if shear limitations can be overcome, at least a 30-fold improvement in productivity should be possible. This is the challenge b This represents 25% utilization of O2 in sparged air. c This represents 20% of saturation dissolved oxygen level. 

Mechanically agitated reactors have been used for cultivation of plant cells (Kato et al., 1972 Tanaka, 1981). Ajar fermenter with a six fiat-blade turbine and a modified paddle has been used by Tanaka (1981) and a similar jar fermenter with two disk turbine impellers has been used by Kato et al. (1972) at about 50 rpm with no significant shear damage to plant cells. Paddle-type impellers were found to be more appropriate (less shear damage) than flat-blade turbine type impellers (Kato et al., 1972). The only production-scale reactor used for shikonin production in Japan is also an agitated vessel. 

DiCosmo and Misawa (14) suggested the immense potential of plant cell culture- elicitor (inducer) interactions to the large scale production of secondary metabolites with the induction of shikonin formation by agar in Lithospermum erythrorhizon cell suspension cultures (39) this is so far one of the most successful examples of elicitor effects. Some reports on the induction of enzymes of plant... 

Plant derived pharmaceuticals are estimated to have an annual value of 9 billion in the U.S. alone (4). Flavors and fragrances have a current worldwide market of about 1.5 billion. Market data for insecticides and other fine chemicals such as pigments are not readily available. The first example, and the only current commercial process based on plant cell culture, is for the production of shikonin in Japan. This compound is both used in medicine and as a pigment (5-7. ... 

The first plant product commercially produced by plant cell culture was the prenylated anthraquinone shikonin 16, from the boraginaceous plant Lithospermum erythrorhizon Sieb. et Zucc. (Mitsui Petrochemical Industry Company) in 1983.25 Shikonin is used as a dye in cosmetics (lipsticks, soaps and lotions) and its production yield from cell cultures was over ten-fold its isolation yield from the intact plant.25 In practice, eight runs of two weeks each in a 200 L bioreactor could afford the amount of shikonin produced in four years by a 1 ha field of L. erythrorhizon 25 Shikonin has an interesting and pleiotropic biological profile, which includes insulin mimicry and interference with protein-protein interactions, but it has not yet found medicinal application.26... 

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