Keizo Hosokawa

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.

GENETIC TRANSFORMATION OF GENTIAN USING WILD-TYPE AGROBACTERIUM RHIZOGENES

Agrobacterium rhizogenes A4 harboring the agropine type root-inducing plasmid (pRiA4) induced hairy roots on stem sections of gentian plants. Transgenic gential plants were regenerated from the hairy roots at a frequency of 19% adventitious shoot regeneration on Murashige and Skoog medium containing 30 g l-1 sucrose, 10 mg l-1 N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg l-1 1-naphthaleneacetic acid and 2 g l-1 gellan gum. The shoots were grown to maturity in a growth chamber after acclimatization. The mature transformed plants showed distinct variations in their phenotypes, such as dwarfness. Polymerase chain reaction and Southern blot analysis revealed stable integration of transferred DNA.

Flavonol Caffeoylglycosides as α-Glucosidase Inhibitors from Spiraea cantoniensis Flower

In the screening experiments for rat intestinal alpha-glucosidase inhibitors in 218 plants cultivated in the Japanese temperate region, potent maltase-inhibiting activity was found in the extract of flowers of Spiraea cantoniensis. The enzyme assay guided fractionation of the extract led to the isolation of three flavonol caffeoylglycosides, quercetin 3- O-(6- O-caffeoyl)-beta-galactoside ( 1), kaempferol 3- O-(6- O-caffeoyl)-beta-galactoside ( 2), and kaempferol 3- O-(6- O-caffeoyl)-beta-glucoside ( 3), as rat intestinal maltase inhibitors. This is the first report on the alpha-glucosidase-inhibitory activity of those flavonol caffeoylglycosides. Comparison in the activity of the isolates indicated the importance of caffeoyl substructures in the molecule for the alpha-glucosidase-inhibiting activity. The relatively high contents of the active isolates in the plant suggest that S. cantoniensis could be physiologically useful for treatment of diabetes.

Seven acylated anthocyanins in blue flowers of Gentiana

Abstract Three novel anthocyanins, delphinidin 3,3′- di -O-β- d -glucoside -5-O-(6-O- caffeoyl -β- d -glucoside ) , delphinidin 3,3′- di -O-β- d -glucoside -5-O-(6-O-trans-p- coumaroyl -β- d -glucoside ) and delphinidin 3-O-β- d -glucoside -5-O-(6-O-trans-p- coumaroyl -β- d -glucoside ) , named albireodelphin A, B and C, respectively, were isolated from the blue flowers of Gentiana cv. Albireo, along with two known anthocyanins, gentiodelphin and gentiocyanin A. The complete structure of each anthocyanin was unambiguously determined by one- and two-dimensional NMR and other spectral methods. Two other novel anthocyanins, delphinidin 3-O-β- d -glucoside -5-O-(6-O- caffeoyl -β- d - glucoside )-3′-O-(6-O-trans-p- coumaroyl -β- d -glucoside ) and dephinidin 3-O-β- d -glucoside -5-O-(6-O-trans-p- coumaroyl -β- d -glucoside )-3′-O-(6-O- caffeoyl -β- d -glucoside ) , named albireodelphin D and E, respectively, were obtained as a mixture and the structures estimated by one- and two-dimensional NMR and other spectral methods.

Seven acylated anthocyanins in the blue flowers of Hyacinthus orientalis

Two novel anthocyanins, delphinidin 3-O-(6-O-cis-p-coumaroyl-beta-D-glucoside)-5-O-(6-O-malonyl-beta-D -glucoside) and petunidin 3-O-(6-O-trans-p-coumaroyl-beta-D-glucoside)-5-O-(6-O-malonyl-beta -D-glucoside) , were isolated from the blue flowers of Hyacinthus orientalis cv. Delft Blue. Five known acylated anthocyanins, namely, delphinidin 3-O-(6-O-trans-p-coumaroyl-beta-D-glucoside)-5-O-(6-O-beta-D-gluco side), delphinidin 3-O-(6-O-trans-caffeoyl-beta-D-glucoside)-5-O-(6-O-malonyl-beta-D- glucoside), delphinidin 3-O-(6-O-trans-p-coumaroyl-beta-D-glucoside)-5-O-(6-O-malonyl-beta -D-glucoside) , cyanidin 3-O-(6-O-trans-p-coumaroyl-beta-D-glucoside)-5-O-(6-O-malonyl-beta -D-glucoside) and pelargonidin 3-O-(6-O-trans-p-coumaroyl-beta-D-glucoside)-5-O-(6-O-malonyl-beta -D-glucoside) , were also isolated from these flowers. The complete structure of each compound was unambiguously determined by one- and two-dimensional NMR techniques.

Three acylated cyanidin glucosides in pink flowers of gentiana

Abstract Three novel anthocyanins, cyanidin 3-O-β- d -glucoside -5-O-(6-O- caffeoyl -β- d -glucoside ) , cyanidin 3-O-β- d -glucoside -5,3′- bis -O-(6-O- caffeoyl -β- d -glucoside ) and cyanidin 3-O-β- d -glucoside -5-O-(6-O-trans-p- coumaroyl -β- d -glucoside ) , gentiocyanin A, B and C, respectively, were isolated from pink flowers of gentian, along with a known anthocyanin, cyanidin 3-O-β- d -glucoside . The complete structure of each anthocyanin was unambiguously determined by 1D- and 2D-NMR and other spectral methods.

Plant regeneration from protoplasts of Gentiana by embedding protoplasts in gellan gum

A protoplast-to-plant system was developed in Gentiana using a gellan gum-embedding culture. Viable protoplasts could be routinely isolated from in vitro-grown plantlets, and they were embedded in 0.2% gellan gum-solidified B5 medium containing 2 mg l-1 NAA, 0.1 mg l-1 TDZ, 0.1 M sucrose and 0.4 M mannitol. Weekly addition of fresh liquid medium was essential for preventing cell browning. Colony growth was promoted by lowering mannitol concentration of the culture media after one month, and visible colonies were produced after 2 months of culture. Shoot regeneration from protoplast-derived calluses was stimulated by 1 to 10 mg l-1 TDZ in combination with 0.1 mg l-1 NAA. Protoplast-derived plants were recovered following rooting of the shoots in plant growth regulator-free medium and they were successfully transferred to soil.

Acylated anthocyanins from red Hyacinthus orientalis

Abstract Two novel anthocyanins, pelargonidin 3-O-β- d -glucoside -5-O-(6-O- malonyl -β- d -glucoside ) and pelargonidin 3-O-(6-O-trans-p- coumaroyl -β- d -glucoside )-5-O-(4-O- malonyl -β- d -glucoside ) have been isolated from red flowers of Hyacinthus orientalis cv Holly Hock, along with seven known anthocyanins, two of which include cis - p -coumaric acid as the acyl moiety. Their complete structures were unambiguously elucidated by 1D and 2D NMR techniques and other spectral evidence.

Methyl Caffeate as an α-Glucosidase Inhibitor from Solanum torvum Fruits and the Activity of Related Compounds

In screening experiments for rat intestinal α-glucosidase (sucrase and maltase) inhibitors in 325 plants cultivated in Japan’s southern island, of Tanegashima, marked inhibition against both sucrase and maltase was found in the extract of the fruit of Solanum torvum. Enzyme-assay guided fractionation of the extract led to the isolation of methyl caffeate (1) as a rat intestinal sucrase and maltase inhibitor. We examined 13 caffeoyl derivatives for sucrase- and maltase-inhibitory activities. The results showed that methyl caffeate (1) had a most favorable structure for both sucrase and maltase inhibition, except for a higher activity of methyl 3,4,5-trihydroxycinnamate (14) against sucrase. Its moderate inhibitory action against α-glucosidase provides a prospect for antidiabetic usage of S. torvum fruit.

Production of acylated anthocyanins by blue flowers of Hyacinthus orient alis regenerated in vitro

Abstract Acylated anthocyanins were produced by flowers of Hyacinthus orientalis that had been regenerated in vitro . The anthocyanin production was affected by the concentration of gibberellic acid (GA 3 ) and sucrose in Murashige and Skoogs (MS) medium and by temperature. The highest concentration of anthocyanin was obtained when the regenerated flowers were cultured for three weeks at 15° on MS medium containing 1 mgl −1 GA 3 and 30 gl −1 sucrose. The concentration of anthocyanin in the regenerated flowers was ca 1.2 times higher than that in field-grown flowers. The anthocyanins in the regenerated flowers were compared with those in field-grown flowers by chromatographic and spectroscopic methods, showing that these flowers produce almost the same anthocyanins with a little difference in composition.