Shikonin erythrorhizon

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. 

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. 

Shikonin (Cl 75535) occurs as an acetyl derivative in the Japanese sliikone, Lithospermum erythrorhizon, another member of the Boraginaceae family. It is the (fi)-optical isomer of alkannin, Tissue cultures of L en-throrhizon are used in Japan to manufacture shikonin mainly for cosmetic use. Both alkannin and shikonin are mordant dyes producing violet to gray colors on fabrics. Shikalkin the racemate, has been synthesized. 

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. 

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. 

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... 

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... 

Shikonin (= 1 7 -isomer of Alkannin) (naphthoquinone) Echium lycopsis, Lithospermum erythrorhizon [root], Onosma caucaskum (Boraginaceae) TOPI (TOPII) [red colour]... 

Lange, B.M., Severin, K., Bechthold, A. and Heide, L. (1998a) Regulatory role of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase for shikonin biosynthesis in Lithospermum erythrorhizon cell suspension cultures. Planta, 204, 234- 1. 

An additional insight into the importance of specific media components on production of secondary products can be gained by examining the case history of shikonin production. It had been shown that callus cultures of Lithospermum erythrorhizon could be induced to produce shikonin on Linsmaier-Skoog medium supplemented with lpM indole acetic acid (IAA) and lOpM kinetin (KIN) (52). The effects of specific nutritional components of the tissue culture medium on growth of the cell cultures and production of shikonin were also investigated i53). Fujita et al. (54,55) found that the levels of NO,, Cu+, and SO had profound effects of shikonin biosynthesis. Optimal concentrations were identified for each ion (I 8 ) as well as optimal levels of key organic components. The resultant medium supported production of shikonin at a rate approximately 13 times that obtained on previous media formulations. 

For shikonin production hy Lithospermum erythrorhizon, two-stage cell culture was used (see Fig. 28). The first stage culture was grown in a MG-5 medium which was suitable for cell mass production. It was then transferred to 2nd-stage culture where it was grown in an M-9 medium, modified by a higher Cu" content and a decreased salt content. 

Successful commercialisation of the formation of the secondary compounds by plant cell cultures has been achieved by the production of shikonin pigments from plant cell cultures of Lithospermun erythrorhizon S. Hwang by Mitsui Petrochemical Industries Ltd. Several studies have investigated factors influencing shikonin production in cultured cells. For example, studies were conducted with a BK-39 callus culture of Lithospermun erythrorhizon, which produced seven shikonin derivatives. Selected BK-39 cultures produced almost the same profile of shikonin naphthoquinones as the initial culture [352]. 

Extracts from the root of Ko-shikon (Lithospermum erythrorhizon) have long been used in Japan as dyes and for their anti-bacterial activity in the treatment of wounds, and burns. The active principles, a series of anthraqmnone derivatives known as shikonins, accumulate in the root to a level of 2% (dry wt.). The total supply of Ko-shikon is imported from China and Korea and it takes the plants 5-7 years to grow to commercial size. Pure natural shikonin sells for about 4500/kg. Based on an earlier report showing shikonin production in root derived callus cultures... 

Hisa, T., Kimura, Y., Takada, K., Suzuki, F., and Takigawa, M., Shikonin, an ingredient of Lithospermum erythrorhizon, inhibits angiogenesis in vivo and in vitro. Anticancer Res., 18, 783-790, 1998. 

Fukui, H., Tani, M., and Tabata, M. 1990. Induction of shikonin biosynthesis by endogenous polysaccharides in Lithospermum polysaccharides in Lithospermum erythrorhizon cell suspension cultures. Plant Cell Rep., 9 73-76. 

Prenylated naphthoquinones occur in the roots of several genera of the Boraginaceae (Leistner, 1985). The shikonins (55), an important group of prenylated naphthoquinones used in Japan for their anti-inflammatory properties, antibacterial activity, and as dyestuffs, have become one of the most important groups of secondary compounds produced in tissue culture (Ellis, 1988 Flores et al, 1987). These compounds come from the roots of Lithospermum erythrorhizon (Boraginaceae). Although the roots accumulate up to 2% naphthoquinones, several years are required for the plant to reach commercial size. In a 23-day fermentation period, cells in a 750-L tank accumulated 23% of their dry weight as shikonins (Flores et al, 1987). 

Yazaki, K., Kunihisa, M., Fujisaki, T. Sato, F. (2002). Geranyl diphosphate 4-hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon cloning and characterization of a key enzyme in shikonin biosynthesis. Journal of Biological Chemistry, 277, 6240-6246. 

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