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Hajime Mizukami - One of the best experts on this subject based on the ideXlab platform.
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CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4'-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis.
FEBS letters, 2002Co-Authors: Michiyo Matsuno, Y. Ogihara, Brian E. Ellis, Akito Nagatsu, Hajime MizukamiAbstract:Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4'-hydroxyphenyllactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid biosynthesis.
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cyp98a6 from Lithospermum erythrorhizon encodes 4 coumaroyl 4 hydroxyphenyllactic acid 3 hydroxylase involved in rosmarinic acid biosynthesis
FEBS Letters, 2002Co-Authors: Michiyo Matsuno, Y. Ogihara, Brian E. Ellis, Akito Nagatsu, Hajime MizukamiAbstract:Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4P-hydroxyphenyl- lactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid bio- synthesis. fl 2002 Federation of European Biochemical Soci- eties. Published by Elsevier Science B.V. All rights reserved.
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Cinnamic acid 4-hydroxylase from Lithospermum erythrorhizon: cDNA cloning and gene expression
Plant Cell Reports, 2001Co-Authors: Y. Yamamura, Y. Ogihara, Hajime MizukamiAbstract:Two near full-length cDNA clones (LEC4H-1 and LEC4H-2) encoding plant cytochrome P450 were isolated by screening a cDNA library constructed from shikonin-producing Lithospermum erythrorhizon callus cultures. The deduced amino acid sequences of LEC4H-1 and LEC4H-2 exhibited 91% identity and were highly homologous to various cinnamic acid 4-hydroxylase (C4H) sequences from higher plants. Expression studies in yeast confirmed that the P450 clones encode C4H. The level of C4H transcripts in the cultured cells was unaffected by environmental factors, including light irradiation, supply of auxin and methyl jasmonate addition, indicating that transcription of these genes is constitutive and not involved in the regulation of shikonin and rosmarinic acid biosynthesis by these factors.
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Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures
Plant Cell Reports, 1993Co-Authors: Hajime Mizukami, Yukari Tabira, Brian E. EllisAbstract:A dramatic increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after their exposure to methyl jasmonate (MJ). Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) and 4-hydroxyphenylpyruvate reductase (HPR) activities increased rapidly and transiently, whereas tyrosine aminotransferase (TAT) activity showed only a slight increase. The elicitation activity of MJ was much higher than that of yeast extract (YE) in terms of the induction of PAL and HPR activities, RA accumulation and incorporation of both ^14C-phenylalanine and ^14C-tyrosine into RA. However, the response of the cultured cells to MJ-treatment was slower than that to YE-treatment.
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Induction of rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures by yeast extract.
Plant cell reports, 1992Co-Authors: Hajime Mizukami, Terumi Ogawa, Hiromu Ohashi, Brian E. EllisAbstract:A transient increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after addition of yeast extract (YE) to the suspension cultures, reaching a maximum at 24 hr. The highest increase of the RA content (2.5-fold) was obtained when 6-day-old cells in the exponential growth phase were treated with YE. Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) activity increased rapidly, whereas tyrosine aminotransferase (TAT) activity was largely unaffected by the treatment. The incorporation of both 14C-phenylalanine and 14C-tyrosine into RA was enhanced in the YE-treated cells, consistent with increased synthesis of the ester.
Lutz Heide - One of the best experts on this subject based on the ideXlab platform.
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4-Hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon: Purification of a plant membrane-bound prenyltransferase
Planta, 1998Co-Authors: Agnes Muhlenweg, Martin Melzer, Shu-ming Li, Lutz HeideAbstract:Geranyldiphosphate:4-hydroxybenzoate 3-geranyltransferase is a regulatory enzyme in the biosynthesis of shikonin, a phytoalexin and pharmaceutical produced by cell cultures of Lithospermum erythrorhizon Sieb. et Zucc. In Linsmaier-Skoog medium, the activity of this enzyme could be enhanced more than 200-fold by addition of methyl jasmonate, and this culture material was used for the solubilization and purification of the enzyme. Of various detergents examined, digitonin was the most suitable for the solubilization of the enzyme. The solubilized enzyme was purified 800-fold by chromatography over diethylaminoethyl (DEAE)-Sephacel, Heparin-Sepharose, Reactive Green 19-Agarose, and Cholic Acid-Agarose. The purified enzyme required magnesium ions as cofactor and was highly specific for geranyldiphosphate (GPP) and 4-hydroxybenzoate (4HB) as substrates. The K(m) values for 4HB and GPP were calculated by the method of Lineweaver and Burk as 18.4 microM and 13.8 microM, respectively.
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Purification of UDP-glucose: 4-hydroxybenzoate glucosyltransferase from cell cultures of Lithospermum erythrorhizon
Phytochemistry, 1997Co-Authors: Zhao-xin Wang, Lutz HeideAbstract:Abstract UDP-glucose: 4-hydroxybenzoate glucosyltransferase (4HB glucosyltransferase, EC 2.4.1.194) was purified to near homogeneity from cell suspension cultures of Lithospermum erythrorhizon , using ammonium sulphate precipitation, and column chromatography on DEAE Sephacel, Superdex 200, hydroxyapatite, HiTrap blue, Mono P and Mono Q. Affinity chromatography on HiTrap blue was the crucial step for the success of this scheme. After removal of NAD-dependent enzymes from the HiTrap blue column with NAD (5 mM), the 4HB glucosyltransferase was eluted specifically with UDP-glucose (0–20 mM). The above purification procedure resulted in an enzyme fraction with 27 300-fold higher specific 4HB glucosyltransferase activity than the crude enzyme extract. The enzyme is a monomer of about M r , 51 k, determined by SDS-PAGE. © 1997 Published by Elsevier Science Ltd. All rights reserved
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cDNA Cloning and Gene Expression of Phenylalanine Ammonia-Lyase in Lithospermum erythrorhizon
Bioscience biotechnology and biochemistry, 1997Co-Authors: Kazufumi Yazaki, Klaus Severin, Mieko Kataoka, Gisho Honda, Lutz HeideAbstract:Two cDNA clones (LEPAL-1 and LEPAL-2) encoding phenylalanine ammonia-lyase were isolated from cell suspension cultures of Lithospermum erythrorhizon. Northern kinetic studies showed that LEPAL-1 mRNA contents markedly increased one day after inoculation of the cells into fresh medium, then decreased to the steady-state level. The course of mRNA accumulation paralleled that of PAL enzyme activity. The rapid induction of PAL activity seems to reflect the induction of dihydroechinofuran biosynthesis, while shikonin was produced at the steady-state level of PAL activity. The course of LEPAL-2 mRNA accumulation seemed to be similar to, but much lower than that of LEPAL-1. In the intact plant, both genes are expressed mainly in the root, the organ in which shikonin is exclusively produced and accumulated. Genomic Southern blot analyses showed that both genes are present in the genome as single copies.
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4-hydroxybenzoate prenyltransferases in cell-free extracts of Lithospermum erythrorhizon cell cultures
Phytochemistry, 1997Co-Authors: Robert Boehm, Martin Melzer, Lutz HeideAbstract:Abstract Aromatic prenylation reactions of 4-hydroxybenzoate (4HB) are involved in the biosynthesis of ubiquinones and of shikonin, a naphthoquinone pigment derived from 4HB and geranyldiphosphate (GPP) in Lithospermum erythrorhizon. The enzymic prenylation of 4HB with GPP and with solanesyldiphosphate (SPP) was measured in cell-free extracts from L. erythrorhizon cell cultures. The conversion of GPP was induced by methyl jasmonate, an inducer of shikonin biosynthesis, whilst the conversion of SPP was not, suggesting that the two reactions are carried out by different enzymes. Either reaction was found both in the microsomal fraction and in the organellar membrane fraction. The activity of the 4HB geranyltransferase in the microsomal fraction could be separated from the 4HB solanesyltransferase by partial purification using DEAE Sephacel and Heparin Sepharose affinity columns. The results indicate the presence of two distinct enzymes, GPP:4-HB geranyltransferase and SPP:4-HB solanesyltransferase. The enzymes are apparently quite specific for the chain length of the isoprenoid precursors, which is in contrast to the broad substrate specificity of the polyprenyldiphosphate: 4-HB polyprenyltransferase from Escherichia coli.
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Purification and characterization of an alcohol dehydrogenase from Lithospermum erythrorhizon cell cultures.
Plant cell reports, 1996Co-Authors: Zhao-xin Wang, Lutz HeideAbstract:An NAD-dependent alcohol dehydrogenase has been purified to apparent homogeneity from cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), using protamine sulphate and ammonium sulphate precipitation and chromatography on DEAE-Sephacel, Superdex 200, hydroxyapatite and HiTrap blue. The enzyme is a homodimer with a Mr of ca. 77,000. Each subunit with a Mr of 40,000 contains two zinc atoms. Its isoelectric point was found at pH 5.0. The best alcohol substrate of the enzyme is ethanol. The pH optimum for ethanol oxidation is at pH 8.7 and for acetaldehyde reduction at pH 4.6. The Michaelis constants for ethanol and NAD are 2.49 and 0.05 (pH 8.7), and for acetaldehyde and NADH 2.2 and 0.078 mM (pH 4.6), respectively. Partial amino acid sequences of the purified enzyme showed high homology to alcohol dehydrogenases from other plants.
Koichiro Shimomura - One of the best experts on this subject based on the ideXlab platform.
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Ethylene induced shikonin biosynthesis in shoot culture of Lithospermum erythrorhizon.
Plant physiology and biochemistry : PPB, 2005Co-Authors: Kaori Touno, Jin Tamaoka, Yuko Ohashi, Koichiro ShimomuraAbstract:Lithospermum erythrorhizon shoots, cultured on phytohormone-free Murashige and Skoog solid medium, produced shikonin derivatives, whereas shoots cultured in well-ventilated petri dishes, produced small amount. Analysis by gas chromatography revealed the presence of ethylene in non-ventilated petri dishes where the shoots, producing shikonin derivatives, were cultured. Therefore, the possible involvement of ethylene in shikonin biosynthesis of shoot cultures was investigated. Treatment of ethylene or the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, resulted in increasing shikonin derivatives contents in cultured shoots. Silver ion, an ethylene-response inhibitor, or aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, decreased production of shikonin derivatives in cultured shoots. Our results indicate that ethylene is one of the regulatory elements of shikonin biosynthesis in L. erythrorhizon shoot culture.
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Histological Observation of Red Pigment Formed on Shoot Stem of Lithospermum erythrorhizon
Plant Biotechnology, 2000Co-Authors: Kaori Touno, Kazufumi Yazaki, Kyuya Harada, Kayo Yoshimatsu, Koichiro ShimomuraAbstract:Shikonin production on the stem of shoot cultures of Lithospermum erythrorhizon was controlled by use of various culture media and light irradiation. Microscopic analysis of the shoot cultures revealed that the red pigment formation was only observed on the stem surface and the stem hairs of shoots cultured in the dark. Cross section of the shoot stem which formed red pigment in the dark was morphologically similar to that of shoots cultured under illumination. Red pigment accumulation was strictly localized in the outer surface of epidermal cells. The localization of these pigments was similarly observed in root tissues generated from the cultured shoots as well as field-grown roots. Northern blot analysis indicated that LEDI-2 gene, which is one of the candidates for the regulatory element of shikonin biosynthesis and specifically expressed in the root system of the intact plants, was also expressed in the stem of shoot cultures when producing shikonin.
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Shikonin production and secretion by hairy root cultures of Lithospermum erythrorhizon
Plant Cell Reports, 1991Co-Authors: Koichiro Shimomura, Hiroshi Sudo, Hitoshi Saga, Hiroshi KamadaAbstract: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.
Brian E. Ellis - One of the best experts on this subject based on the ideXlab platform.
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CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4'-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis.
FEBS letters, 2002Co-Authors: Michiyo Matsuno, Y. Ogihara, Brian E. Ellis, Akito Nagatsu, Hajime MizukamiAbstract:Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4'-hydroxyphenyllactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid biosynthesis.
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cyp98a6 from Lithospermum erythrorhizon encodes 4 coumaroyl 4 hydroxyphenyllactic acid 3 hydroxylase involved in rosmarinic acid biosynthesis
FEBS Letters, 2002Co-Authors: Michiyo Matsuno, Y. Ogihara, Brian E. Ellis, Akito Nagatsu, Hajime MizukamiAbstract:Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4P-hydroxyphenyl- lactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid bio- synthesis. fl 2002 Federation of European Biochemical Soci- eties. Published by Elsevier Science B.V. All rights reserved.
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Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures
Plant Cell Reports, 1993Co-Authors: Hajime Mizukami, Yukari Tabira, Brian E. EllisAbstract:A dramatic increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after their exposure to methyl jasmonate (MJ). Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) and 4-hydroxyphenylpyruvate reductase (HPR) activities increased rapidly and transiently, whereas tyrosine aminotransferase (TAT) activity showed only a slight increase. The elicitation activity of MJ was much higher than that of yeast extract (YE) in terms of the induction of PAL and HPR activities, RA accumulation and incorporation of both ^14C-phenylalanine and ^14C-tyrosine into RA. However, the response of the cultured cells to MJ-treatment was slower than that to YE-treatment.
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Induction of rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures by yeast extract.
Plant cell reports, 1992Co-Authors: Hajime Mizukami, Terumi Ogawa, Hiromu Ohashi, Brian E. EllisAbstract:A transient increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after addition of yeast extract (YE) to the suspension cultures, reaching a maximum at 24 hr. The highest increase of the RA content (2.5-fold) was obtained when 6-day-old cells in the exponential growth phase were treated with YE. Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) activity increased rapidly, whereas tyrosine aminotransferase (TAT) activity was largely unaffected by the treatment. The incorporation of both 14C-phenylalanine and 14C-tyrosine into RA was enhanced in the YE-treated cells, consistent with increased synthesis of the ester.
Kazufumi Yazaki - One of the best experts on this subject based on the ideXlab platform.
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Lithospermum erythrorhizon cell cultures: Present and future aspects.
Plant biotechnology (Tokyo Japan), 2017Co-Authors: Kazufumi YazakiAbstract: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.
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Cell Development during Somatic Embryogenesis in Lithospermum erythrorhizon Observed by Scanning Electron Microscopy
Asian Journal of Applied Sciences, 2015Co-Authors: Totik Sri Mariani, Kazufumi Yazaki, Hiroshi MiyakeAbstract:Scanning electron microscopy observation could be used to describe cell development during somatic embryogenesis in Lithospermum erythrorhizon. The purpose of this study is to provide numbers of important morphological information in order to understand the embryogenesis of this plant. Proembryo was composed of small round cells. Proliferating embryogenic cell (PEC) was composed of small round cells in the center, surrounded by slightly elongated cells at the periphery. Globular somatic embryo composed of small round cells. Its surface was covered by extracellular material. Heart somatic embryo was composed of small round cells. Slightly elongated cells in pre-root apex and round cells in pre-shoot apex of early torpedo somatic embryo showing polarization of somatic embryo. In late torpedo somatic embryos, pre-root apex became firm and pre-shoot apex still consisted of round cells.
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geranyl diphosphate 4 hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon cloning and characterization of a ket enzyme in shikonin biosynthesis
Journal of Biological Chemistry, 2002Co-Authors: Kazufumi Yazaki, Miyuki Kunihisa, Takahiro Fujisaki, Fumihiko SatoAbstract:Two cDNAs encoding geranyl diphosphate:4-hy- droxybenzoate 3-geranyltransferase were isolated from Lithospermum erythrorhizon by nested PCR using the conserved amino acid sequences among polyprenyl- transferases for ubiquinone biosynthesis. They were functionally expressed in yeast COQ2 disruptant and showed a strict substrate specificity for geranyl diphosphate as the prenyl donor, in contrast to ubiquinone biosynthetic enzymes, suggesting that they are involved in the biosynthesis of shikonin, a naphthoquinone secondary metabolite. Regulation of their expression by various culture conditions coincided with that of geranyltransferase activity and the secondary metabolites biosynthesized via this enzyme. This is the first established plant prenyltransferase that transfers the prenyl chain to an aromatic substrate.
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Regulation of Lithospermic Acid B and Shikonin Production in Lithospermum erythrorhizon Cell Suspension Cultures
Chemical & pharmaceutical bulletin, 2002Co-Authors: Hirobumi Yamamoto, Kazufumi Yazaki, Ping Zhao, Kenichiro InoueAbstract:Cell suspension cultures of Lithospermum erythrorhizon produced a large amount of lithospermic acid B, a caffeic acid tetramer, as well as shikonin derivatives (each ca. 10% of dry wt.) when cultured in shikonin production medium M-9. Various culture factors for increasing the production of lithospermic acid B were investigated. Lithospermic acid B production was inhibited by 2, 4-D or NH4+, whereas it was stimulated by Cu2+. These regulatory patterns were similar to those for the production of shikonin derivatives in these cell cultures, suggestive of close relations and similar metabolic regulation between the production of these compounds. Cultivation under light illumination, however, showed that these metabolisms were independently regulated. In particular, blue light showed a stimulatory effect on lithospermic acid B production, while shikonin production was strongly inhibited, indicative of an effective condition for lithospermic acid B production.
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Caffeic acid oligomers in Lithospermum erythrorhizon cell suspension cultures.
Phytochemistry, 2000Co-Authors: Hirobumi Yamamoto, Kenichiro Inoue, Kazufumi YazakiAbstract:Abstract Lithospermum erythrorhizon cells cultured in pigment production (M-9) medium produced lithospermic acid B, a dimerized caffeic acid ester derivative, in quantities similar to the production of shikonin. The cells also produced a related dimer, (+)-rabdosiin. In Linsmaier–Skoog liquid medium, which suppresses shikonin production, both lithospermic acid B and (+)-rabdosiin were still formed. Lithospermic acid, a caffeic acid–rosmarinic acid conjugate, was isolated as a main constituent in Lithospermum hairy root cultures. In the aerial parts of L. erythrorhizon, the content of these phenylpropanoid oligomers was relatively low compared to that of rosmarinic acid.