Lithospermum 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. 

Lithospermum erythrorhizon, L. officinalis Adina rubella, A. ratemosa Panax ginseng... 

Lithospermum erythrorhizon, L. officinale Lobelia inflata Lobelia inflata... 

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]. 

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. 

YAZAK1, K., KUNMSHA, M., FUJ1SAKI, T., SATO, F Geranyl diphosphate 4-hydroxybenzoate gerany ltransferase from Lithospermum erythrorhizon. J. Biol. Chem., 2002,277,6240-6246. 

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... 

Another important observation concerning growth characteristics of plant cells deals with the separation of growth and production phases, especially for a large number of secondary metabolities. Certain media components will favor growth while other components will favor secondary metabolite production. This has been extensively documented with pigment formation by Lithospermum erythrorhizon (33) and the phenolics production in Nicotiana tabacum (34). 

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... 

Echium lycopsis, Lithospermum erythrorhizon, Onosma caucasicum (Boraginaceae) Animals leucocyte cytokine... 

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. 

Muhlenweg, A., Melzer, M., Li, S.M. and Heide, L. (1998) 4-Hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon purification of a plant mem-branebound prenyltransferase. Planta, 205, 407-13. 

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. 

Figure 31. Scale up of suspension cultures for Lithospermum erythrorhizon cells. (Fujita andTabata, 1987).

The in vitro conversion of tyrosine (12) (Figure 12) to 4-hydroxyphenylpynivate (10) and its subsequent reduction to 4-hydroxyphenyllactate (43) has been shown in the extracts of rat liver and yeast. Reactions involved in the conversion of 4-hydroxyphenyllactate (43) to 4-HB (11) are not known. However, it has been suggested that 4-hydroxyphenyllactate is converted to 4-hydroxycinnamate (44), followed by /3-oxidation as the CoA derivative, resulting in the formation of 4-HB " (see Figure 12, (44) (45) (46) (48) (1 )) Evidence in support of /3-oxidation has been obtained in tissue cultures of the plant Lithospermum erythrorhizon. ... 

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