Glycosylation of Stilbene Compounds by Cultured Plant Cells.

Oxyresveratrol and gnetol are naturally occurring stilbene compounds, which have diverse pharmacological activities. The water-insolubility of these compounds limits their further pharmacological exploitation. The glycosylation of bioactive compounds can enhance their water-solubility, physicochemical stability, intestinal absorption, and biological half-life, and improve their bio- and pharmacological properties. Plant cell cultures are ideal systems for propagating rare plants and for studying the biosynthesis of secondary metabolites. Furthermore, the biotransformation of various organic compounds has been investigated as a target in the biotechnological application of plant cell culture systems. Cultured plant cells can glycosylate not only endogenous metabolic intermediates but also xenobiotics. In plants, glycosylation reaction acts for decreasing the toxicity of xenobiotics. There have been a few studies of glycosylation of exogenously administrated stilbene compounds at their 3- and 4'-positions by cultured plant cells of Ipomoea batatas and Strophanthus gratus so far. However, little attention has been paid to the glycosylation of 2'-hydroxy group of stilbene compounds by cultured plant cells. In this work, it is described that oxyresveratrol (3,5,2',4'-tetrahydroxystilbene) was transformed to 3-, 2'-, and 4'-ß-glucosides of oxyresveratrol by biotransformation with cultured Phytolacca americana cells. On the other hand, gnetol (3,5,2',6'-tetrahydroxystilbene) was converted into 2'-ß-glucoside of gnetol by cultured P. americana cells. Oxyresveratrol 2'-ß-glucoside and gnetol 2'-ß-glucoside are two new compounds. This paper reports, for the first time, the glycosylation of stilbene compounds at their 2'-position by cultured plant cells.

Supermolecular drug challenge to overcome drug resistance in cancer cells.

Overcoming multidrug resistance (MDR) of cancer cells can be accomplished using drug delivery systems in large-molecular-weight ATP-binding cassette transporters before entry into phagolysosomes and by particle-cell-surface interactions. However, these hypotheses do not address the intratumoral heterogeneity in cancer. Anti-MDR must be related to alterations of drug targets, expression of detoxification, as well as altered proliferation. In this study, it is shown that the excellent efficacy and sustainability of anti-MDR is due to a stable ES complex because of the allosteric facilities of artificial enzymes when they are used as supermolecular complexes. The allosteric effect of supermolecular drugs can be explained by the induced-fit model and can provide stable feedback control systems through the loop transfer function of the Hill equation.

Glycosylation and Methylation of Quercetin and Myricetin by Cultured Cells of Phytolacca americana.

The glycosylation and methylation of quercetin by cultured plant cells of Phytolacca americana gave quercetin 3-Ο-ß-D-glucoside and isorharnnetin 3-Ο-ß-D- glucoside. Myricetin was glycosylated and methylated to syringetin 3-Ο-ß-D-glucoside by cultured P. americana cells.

Synthesis, oxygen radical absorbance capacity, and tyrosinase inhibitory activity of glycosides of resveratrol, pterostilbene, and pinostilbene.

The stilbene compound resveratrol was glycosylated to give its 4'-O-ß-D-glucoside as the major product in addition to its 3-O-ß-D-glucoside by a plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. This enzyme transformed pterostilbene to its 4'-O-ß-D-glucoside, and converted pinostilbene to its 4'-O-ß-D-glucoside as a major product and its 3-O-ß-D-glucoside as a minor product. An analysis of antioxidant capacity showed that the above stilbene glycosides had lower oxygen radical absorbance capacity (ORAC) values than those of the corresponding stilbene aglycones. The 3-O-ß-D-glucoside of resveratrol showed the highest ORAC value among the stilbene glycosides tested, and pinostilbene had the highest value among the stilbene compounds. The tyrosinase inhibitory activities of the stilbene aglycones were improved by glycosylation; the stilbene glycosides had higher activities than the stilbene aglycones. Resveratrol 3-O-ß-D-glucoside had the highest tyrosinase inhibitory activity among the stilbene compounds tested.

Hydroxylation and Glycosylation of Phenylpropanoids by Cultured Cells of Phytolacca americana.

Hydroxylation and glycosylation of cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid were investigated using cultured plant cells of Phytolacca americana as biocatalysts. Regioselective hydroxylation at the 4-position of cinnamic acid and 3-position of p-coumaric acid was observed. Although cinnamic acid was transformed to mono-glucoside products, di-glycosylation occurred in the case of the biotransformation of p-coumaric acid, caffeic acid, and ferulic acid.

Glycosylation of Chrysin by Cultured Cells of Eucalyptus perriniana.

The biotransformation of chrysin was investigated using cultured plant cells of Eucalyptus perriniana as biocatalysts. Chrysin was glucosylated to chrysin 7- 0-ß-D-glucoside and chrysin 7-ß-p-gentiobioside.

Synthesis and pharmacological evaluation of glycosides of resveratrol, pterostilbene, and piceatannol.

To enhance their water solubility and pharmacological activities, the stilbenes resveratrol, pterostilbene, and piceatannol were glycosylated to their monoglucosides (ß-glucosides) and diglycosides (ß-maltosides) by cultured cells and cyclodextrin glucanotransferase (CGTase). Cultured cells of Phytolacca americana and glucosyltransferase (PaGT) were capable of glucosylation of resveratrol to its 3- and 4'-ß-glucosides. Pterostilbene was slightly transformed into its 4'-ß-glucoside by P. americana cells. Piceatannol was readily converted into piceatannol 4'-ß-glucoside, with the highest yield among the three substrates. The 3- and 4'-ß-glucosides of resveratrol were subjected to further glycosylation by CGTase to give 3- and 4'-ß-maltoside derivatives. The inhibitory action of resveratrol and pterostilbene toward histamine release induced with compound 48/80 from rat peritoneal mast cells was improved by ß-glucosylation and/or ß-maltosylation (i.e., the inhibitory activity for histamine release of the 3- and 4'-ß-glucosides of resveratrol, the 3- and 4'-ß-maltosides of resveratrol, and the 4'-ß-glucoside of pterostilbene was higher than that of the corresponding aglycones, resveratrol and pterostilbene, respectively). In addition, the phosphodiesterase (PDE) inhibitory activity of resveratrol and pterostilbene was enhanced by ß-glucosylation and/or ß-maltosylation (i.e., the PDE inhibitory activities of the 3- and 4'-ß-glucosides of resveratrol, the 4'-ß-maltoside of resveratrol, and the 4'-ß-glucoside of pterostilbene were higher than those of the corresponding aglycones, resveratrol and pterostilbene, respectively).

Regioselective Glycosylation of 3-, 5-, 6-, and 7-Hydroxyflavones by Cultured Plant Cells.

Regioselective glycosylation of 3-, 5-, 6-, and 7-hydroxyflavones was investigated using cultured plant cells of Eucalyptus perriniana and Phytolacca americana as biocatalysts. 3- and 7-Hydroxyflavones were practically glycosylated into the corresponding ß-D-glucosides by E. perriniana and P. americana.

Synthesis of Resveratrol Glycosides by Plant Glucosyltransferase and Cyclodextrin Glucanotransferase and Their Neuroprotective Activity.

Resveratrol was converted by glucosyltransferase from Phytolacca americana into its 3- and 4'-O-ß-D-glucosides. On the other hand, further glycosylation of resveratrol 4'-O-ß-D-glucoside by cyclodextrin glucanotransferase gave the 4'-O-ß-maltoside, 4'-O-ß-maltotrioside, 4'-O-ß-maltotetraoside, and 4'-O-ß- maltopentaoside of resveratrol. The six resveratrol glycosides synthesized here showed higher phosphodiesterase inhibitory activity than resveratrol.

Medicinal facilities to B16F10 melanoma cells for distant metastasis control with a supramolecular complex by DEAE-dextran-MMA copolymer/paclitaxel.

The resistance of cancer cells to chemotherapeutic drugs (MDR) is a major problem to be solved. A supramolecular DEAE-dextran-MMA copolymer (DDMC)/paclitaxel (PTX) complex was obtained by using PTX as the guest and DDMC as the host having 50-300 nm in diameter. The drug resistance of B16F10 melanoma cells to paclitaxel was observed, but there is no drug resistance of melanoma cells to the DDMC/PTX complex in vitro. The cell death rate was determined using Michaelis-Menten kinetics, as the DDMC/PTX complex promoted allosteric supramolecular reaction to tubulin. The DDMC/PTX complex showed a very superior anti-cancer activity to paclitaxel alone in vivo. The median survival time (MST) of the saline, PTX, DDMC/PTX4 (particle size, 50 nm), and DDMC/PTX5 (particle size, 290 nm) groups were 120 h (T/C, 1.0), 176 h (T/C, 1.46), 328 h (T/C, 2.73), and 280 h (T/C, 2.33), respectively. The supramolecular DDMC/PTX complex showed the twofold effectiveness of PTX alone (p < 0.036). Histochemical analysis indicated that the administration of DDMC/PTX complex decreased distant metastasis and increased the survival of mice. A mouse of DDMC/PTX4 group in vivo was almost curing after small dermatorrhagia owing to its anti-angiogenesis, and it will be the hemorrhagic necrotic symptom of tumor by the release of "tumor necrosis factor alpha (TNF-α)" cytokine. As the result, the medicinal action of the DDMC/PTX complex will suppress the tumor-associated action of M2 macrophages and will control the metastasis of cancer cells.

Synthesis of glycosides of resveratrol, pterostilbene, and piceatannol, and their anti-oxidant, anti-allergic, and neuroprotective activities.

Resveratrol was glucosylated to its 3- and 4'-ß-glucosides by cultured cells of Phytolacca americana. On the other hand, cultured P. americana cells glucosylated pterostilbene to its 4'-ß-glucoside. P. americana cells converted piceatannol into its 4'-ß-glucoside. The 3- and 4'-ß-glucosides of resveratrol were further glucosylated to 3- and 4'-ß-maltosides of resveratrol, 4'-ß-maltoside of which is a new compound, by cyclodextrin glucanotransferase. Resveratrol 3-ß-glucoside and 3-ß-maltoside showed low 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity, whereas other glucosides had no radical-scavenging activity. Piceatannol 4'-ß-glucoside showed the strongest inhibitory activity among the stilbene glycosides towards histamine release from rat peritoneal mast cells. Pterostilbene 4'-ß-glucoside showed high phosphodiesterase inhibitory activity.

Glycosylation of quercetin with cultured plant cells and cyclodextrin glucanotransferase.

Quercetin was glucosylated by cultured plant cells of lpomoea batatas to its 3- and 7-O-beta-D-glucosides, and 3,7-O-beta-D-diglucoside. On the other hand, further glycosylation of quercetin 3-O-beta-D-glucoside by cyclodextrin glucanotransferase gave the 3-O-beta-maltoside, 3-O-beta-maltotrioside, and 3-O-[beta-maltotetraosides of quercetin.

Glycosylation of artepillin C with cultured plant cells of Phytolacca americana.

Biotransformation of artepillin C was investigated using cultured plant cells as biocatalysts. Artepillin C was converted into its 4- and 9-beta-D-glucosides, and 4,9-beta-D-diglucoside by cultured cells of Phytolacca americana. In contrast, cultured lpomoea batatas cells glucosylated artepillin C to only its 4- and 9-beta-D-glucosides.

Anticancer efficacy of a supramolecular complex of a 2-diethylaminoethyl-dextran-MMA graft copolymer and paclitaxel used as an artificial enzyme.

The anticancer efficacy of a supramolecular complex that was used as an artificial enzyme against multi-drug-resistant cancer cells was confirmed. A complex of diethylaminoethyl-dextran-methacrylic acid methylester copolymer (DDMC)/paclitaxel (PTX), obtained with PTX as the guest and DDMC as the host, formed a nanoparticle 50-300 nm in size. This complex is considered to be useful as a drug delivery system (DDS) for anticancer compounds since it formed a stable polymeric micelle in water. The resistance of B16F10 melanoma cells to PTX was shown clearly through a maximum survival curve. Conversely, the DDMC/PTX complex showed a superior anticancer efficacy and cell killing rate, as determined through a Michaelis-Menten-type equation, which may promote an allosteric supramolecular reaction to tubulin, in the same manner as an enzymatic reaction. The DDMC/PTX complex showed significantly higher anticancer activity compared to PTX alone in mouse skin in vivo. The median survival times of the saline, PTX, DDMC/PTX4 (particle size 50 nm), and DDMC/PTX5 (particle size 290 nm) groups were 120 h (treatment (T)/control (C), 1.0), 176 h (T/C, 1.46), 328 h (T/C, 2.73), and 280 h (T/C, 2.33), respectively. The supramolecular DDMC/PTX complex showed twice the effectiveness of PTX alone (p < 0.036). Above all, the DDMC/PTX complex is not degraded in cells and acts as an intact supramolecular assembly, which adds a new species to the range of DDS.

Regioselective hydroxylation and glucosylation of alpha- and beta-pinenes with cultured cells of Eucalyptus perriniana.

Cultured plant cells of Eucalyptus perriniana regioselectively hydroxylated (+)- and (-)-alpha-pinenes to the corresponding (+)- and (-)-verbenols. In addition, (+)- and (-)-verbenols were converted into mono-beta-D-glucosides. On the other hand, (+)- and (-)-beta-pinenes were transformed into (+)- and (-)-pinocarveol 3-O-beta-D-glucosides via (+)- and (-)-pinocarveols.

Glucosylation of taxifolin with cultured plant cells.

Cultured plant cells of Eucalyptus perriniana glucosylated taxifolin to its 3'- and 7-O-beta-D-glucosides and 3',7-O-beta-D-diglucoside. On the other hand, taxifolin was converted into 3'- and 7-O-beta-D-glucosides by cultured cells of Nicotiana tabacum and Catharanthus roseus.

Glycosylation of vanillin and 8-nordihydrocapsaicin by cultured Eucalyptus perriniana cells.

Glycosylation of vanilloids such as vanillin and 8-nordihydrocapsaicin by cultured plant cells of Eucalyptus perriniana was studied. Vanillin was converted into vanillin 4-O-ß-D-glucopyranoside, vanillyl alcohol, and 4-O-ß-D-glucopyranosylvanillyl alcohol by E. perriniana cells. Incubation of cultured E. perriniana cells with 8-nordihydrocapsaicin gave 8-nordihydrocapsaicin 4-O-ß-D-glucopyranoside and 8-nordihydrocapsaicin 4-O-ß-D-gentiobioside.

Formation of tetrahydrocurcumin by reduction of curcumin with cultured plant cells of Marchantia polymorpha.

Cultured plant cells of Marchantia polymorpha, Nicotiana tabacum, Phytolacca americana, Catharanthus roseus, and Gossypium hirsutum were examined for their ability to reduce curcumin. Only M. polymorpha cells converted curcumin into tetrahydrocurcumin in 90% yield in one day. Time-course experiment revealed a two-step formation of tetrahydrocurcumin via dihydrocurcumin.

Chemo-enzymatic synthesis of ester-linked docetaxel-monosaccharide conjugates as water-soluble prodrugs.

Three new docetaxel prodrugs, i.e., 7-propionyldocetaxel 3''-O-ß-D-glycopyranosides, which contain ester-linked monosaccharides, were synthesized by a chemo-enzymatic procedure involving enzymatic transglycosylations with lactase, ß-galactosidase, or ß-xylosidase. The water-solubility of 7-propionyldocetaxel 3''-O-ß-D-glucopyranoside was 52-fold higher than that of docetaxel. 7-Propionyldocetaxel 3''-O-ß-D-glucopyranoside and 7-propionyldocetaxel 3''-O-ß-D-xylopyranoside were effectively hydrolyzed by the relevant enzyme(s) of human cancer cells to release docetaxel, whereas 7-propionyldocetaxel 3''-O-ß-D-galactopyranoside was relatively resistant under similar conditions. 7-Propionyldocetaxel 3''-O-ß-D-glucopyranoside and 7-propionyldocetaxel 3''-O-ß-D-xylopyranoside showed in vitro cytotoxic activity against human cancer cells, whereas 7-propionyldocetaxel 3''-O-ß-D-galactopyranoside exerted low cytotoxicity.

Biotransformation of cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid by plant cell cultures of Eucalyptus perriniana.

Biotransformations of phenylpropanoids such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid were investigated with plant-cultured cells of Eucalyptus perriniana. The plant-cultured cells of E. perriniana converted cinnamic acid into cinnamic acid ß-D-glucopyranosyl ester, p-coumaric acid, and 4-O-ß-D-glucopyranosylcoumaric acid. p-Coumaric acid was converted into 4-O-ß-D-glucopyranosylcoumaric acid, p-coumaric acid ß-D-glucopyranosyl ester, 4-O-ß-D-glucopyranosylcoumaric acid ß-D-glucopyranosyl ester, a new compound, caffeic acid, and 3-O-ß-D-glucopyranosylcaffeic acid. On the other hand, incubation of caffeic acid with cultured E. perriniana cells gave 3-O-ß-D-glucopyranosylcaffeic acid, 3-O-(6-O-ß-D-glucopyranosyl)-ß-D-glucopyranosylcaffeic acid, a new compound, 3-O-ß-D-glucopyranosylcaffeic acid ß-D-glucopyranosyl ester, 4-O-ß-D-glucopyranosylcaffeic acid, 4-O-ß-D-glucopyranosylcaffeic acid ß-D-glucopyranosyl ester, ferulic acid, and 4-O-ß-D-glucopyranosylferulic acid. 4-O-ß-D-Glucopyranosylferulic acid, ferulic acid ß-D-glucopyranosyl ester, and 4-O-ß-D-glucopyranosylferulic acid ß-D-glucopyranosyl ester were isolated from E. perriniana cells treated with ferulic acid.