Chemical stability and colorant properties of betaxanthin pigments from Celosia argentea.

The chemical stability and colorant properties of three betaxanthins recently identified from Celosia argentea varieties were evaluated. Lyophilized betaxanthin powders from yellow inflorescences of Celosia exhibited bright yellow color and high color purity with strong hygroscopicity. The aqueous solutions containing these betaxanthins were bright yellow in the pH range 2.2-7.0, and they were most stable at pH 5.5. The betaxanthins in a model system (buffer) were susceptible to heat, and found to be as unstable as red betacyanins (betanin and amaranthine) at high temperatures (>40 degrees C), but more stable at 40 degrees C with the exclusion of light and air. The three betaxanthins had slightly higher pigment retention than amaranthine/isoamaranthine in crude extracts at 22 degrees C, as verified by HPLC analysis. Lyophilized betaxanthins had much better storage stability (mean 95.0% pigment retention) than corresponding aqueous solutions (14.8%) at 22 degrees C after 20 weeks. Refrigeration (4 degrees C) significantly increased pigment retention of aqueous betaxanthins to 75.5%.

Betalains of Celosia argentea.

The betalains of yellow, orange and red inflorescences of common cockscomb (Celosia argentea var. cristata) were compared and proved to be qualitatively identical to those of feathered amaranth (Celosia argentea var. plumosa). In addition to the known compounds amaranthin and betalamic acid, the structures of three yellow pigments were elucidated to be immonium conjugates of betalamic acid with dopamine, 3-methoxytyramine and (S)-tryptophan by various spectroscopic techniques and comparison to synthesized reference compounds; the latter two are new to plants. Among the betacyanins occurring in yellow inflorescences in trace amounts, the presence of 2-descarboxy-betanidin, a dopamine-derived betacyanin, has been ascertained. The detection of high dopamine concentration may be of toxicological relevance in use of yellow inflorescences as a vegetable and in traditional Chinese medicine, common uses for the red inflorescences of common cockscomb.

Formation and occurrence of dopamine-derived betacyanins.

In light of the fact that the main betaxanthin (miraxanthin V) and the major betacyanin (2-descarboxy-betanidin) in hairy root cultures of yellow beet (Beta vulgaris L.) are both dopamine-derived, the occurrence of similar structures for the minor betacyanins was also suggested. By HPLC comparison with the betacyanins obtained by dopamine administration to beet seedlings, enzymatic hydrolysis, LCMS and 1H NMR analyses, the minor betacyanins from hairy roots were identified as 2-descarboxy-betanin and its 6'-O-malonyl derivative. A short-term dopamine administration experiment with fodder beet seedlings revealed that the condensation step between 2-descarboxy-cyclo-Dopa and betalamic acid is the decisive reaction, followed by glucosylation and acylation. From these data a pathway for the biosynthesis of dopamine-derived betalains is proposed. Furthermore, the occurrence of these compounds in various cell and hairy root cultures as well as beet plants (Fodder and Garden Beet Group) is shown.

Betalains from Christmas cactus.

The presence of 14 betalain pigments have been detected by their characteristic spectral properties in flower petals of Christmas cactus (Schlumbergera x buckleyi). Along with the known vulgaxanthin I, betalamic acid, betanin and phyllocactin (6'-O-malonylbetanin), the structure of a new phyllocactin-derived betacyanin was elucidated by various spectroscopic techniques and carbohydrate analyses as betanidin 5-O-(2'-O-beta-D-apiofuranosyl-6'-O-malonyl)-beta-D-glucopyranosid e. Among the more complex betacyanins occurring in trace amounts, the presence of a new diacylated betacyanin ¿betanidin 5-O-[(5"-O-E-feruloyl)-2'-O-beta-D- apiofuranosyl-6'-O-malonyl)]-beta-D-glucopyranoside¿ has been ascertained. Furthermore, the accumulation of betalains during flower development and their pattern in different organs of the flower has been examined.

The decisive step in betaxanthin biosynthesis is a spontaneous reaction1

Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris "Golden Beet") showed that all amino acids (S- and R-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris "Altamo") plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly including cyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.

Betacyanins from plants and cell cultures of Phytolacca americana.

Betacyanins from cell cultures of Phytolacca americana were characterized and compared with those of the stems and ripening fruits of the plant. Whereas in fruits prebetanin (betanin 6'-O-sulphate) and its isoform predominate, in the stem and cell cultures feruloylated derivatives occur as the major components. These were rigorously identified by various spectroscopic techniques (DAD-HPLC, NMR, LC-MS and electrospray MS-MS) and carbohydrate analyses as betanidin 5-O-[(5"-O-E-feruloyl)-2'-O-beta-D-apiofuranosyl] -beta-D-glucopyranoside, a new betacyanin of higher plants, and betanidin 5-O-(6'-O-E-feruloyl)-beta-D-glucopyranoside (lampranthin II), together with their isoforms.

Assay for tyrosine hydroxylation activity of tyrosinase from betalain-forming plants and cell cultures.

In our studies on tyrosinase-catalyzed tyrosine hydroxylation, possibly involved in betalain biosynthesis, we have evaluated different assays for the detection and quantification of the enzymatic product Dopa with respect to sensitivity, simplicity, and suitability for automatization. A tyrosinase assay including reversed-phase high-performance liquid chromatography with isocratic elution and fluorescence detection has been developed (native fluorescence of Dopa; excitation at 281 nm, emission at 314 nm). This improved assay was sensitive (detection limit: 2 pmol Dopa) and showed a wide linear range of Dopa detection (10 pmol-20 nmol Dopa). The method proved to be suitable for high-performance liquid chromatography with an autosampler and has been applied for measuring tyrosinase activity of cell cultures and different tissues of Portulaca grandiflora.

Biotransformation of a [14CH3]-2-Methylaminobenzophenone by Plant Cell Cultures.

2-[ (14)C]-Methylamino-2',4'-dimethoxy-6'-hydroxybenzophenone ( 4) was synthesized and administered to RUTA GRAVEOLENS cell suspension cultures. Compound 4 was not incorporated into acridone alkaloids but glucosylated giving 7. This reaction takes place also in cell suspension cultures of ADHATODA VASICA and PEGANUM HARMALA.

Hydrolysis of Conjugated Gibberellins by ß-Glucosidases from Dwarf Rice (Oryza sativa L. cv. «Tan-ginbozu¼).

Dwarf rice (Oryza sativa L. cv. «Tan-ginbozu¼) is widely used for gibberellin conjugate bioassay. Soluble and particulate fractions from seeds and seedlings of this variety showed hydrolytic activity toward [(3)H]GA-O-Glc. The soluble fraction from mature seeds exhibited the highest [(3)H]GA-O-Glc/pNP-ß-Glc hydrolysis ratio which decreased during the early germination phase. The soluble ß-glucosidases from mature seeds were efficiently separated by CMSephadex C-50 column chromatography. It was found that the most active ß-glucosidase CM 1 from seeds hydrolyses GA(8)-O-2-Glc 8 times faster than GA(3)-O-3-Glc and GA(3) Glc ester. In shoots of 4-d-old etiolated seedlings a soluble ß-glucosidase (CM 4) is present which showed a 200 times faster hydrolysis of GA(8)-O-2-Glc as compared to GA(3)-O-3-Glc. Both the ß-glucosidase CM 4 and the main ß-glucosidase component CM 5 possessed relatively high GA(3) Glc ester hydrolysing activity. Our enzymatic findings demonstrate that in dwarf rice bioassay (leaf application) the GA-OGlc with equatorially arranged glucosyl residues (O-2-glucosides of GA(8), GA(26), GA(27), and GA(29)) and GA Glc esters are hydrolysed by the concerted action of the endogenous a-glucosidases CM 4 and CM 5 from shoots.