Regioselective hydroxylation and dehydrogenation of capsaicin and dihydrocapsaicin by cultured cells of Phytolacca americana.

The biotransformations of capsaicin and dihydrocapsaicin were investigated using cultured plant cells of Phytolacca americana as biocatalysts. Four products, ie 15-hydroxycapsaicin, dihydrocapsaicin, 15-hydroxydihydrocapsaicin, and capsaicin 4-ß-glucoside, were isolated from the suspension cultures of P. americana treated with capsaicin for 3 days, showing that capsaicin was regioselectively hydroxylated, reduced, and glucosylated by cultured P. americana cells. On the other hand, dihydrocapsaicin was regioselectively dehydrogenated, hydroxylated, reduced, and glucosylated to give four products, ie capsaicin, 15-hydroxycapsaicin, 15-hydroxydihydrocapsaicin, and capsaicin 4-ß-glucoside, by cultured P. americana cells. In this paper, it is reported, for the first time, that dihydrocapsaicin is converted into 15-hydroxydihydrocapsaicin by plant cultured cells.

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.

Synthesis of Daidzein Glycosides, α-Tocopherol Glycosides, Hesperetin Glycosides by Bioconversion and Their Potential for Anti-Allergic Functional-Foods and Cosmetics.

Daidzein is a common isoflavone, having multiple biological effects such as anti-inflammation, anti-allergy, and anti-aging. α-Tocopherol is the tocopherol isoform with the highest vitamin E activity including anti-allergic activity and anti-cancer activity. Hesperetin is a flavone, which shows potent anti-inflammatory effects. These compounds have shortcomings, i.e., water-insolubility and poor absorption after oral administration. 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. They were transformed by cultured Nicotiana tabacum cells to 7-ß-glucoside and 7-ß-gentiobioside of daidzein, and 3'- and 7-ß-glucosides, 3',7-ß-diglucoside, and 7-ß-gentiobioside of hesperetin. Daidzein and α-tocopherol were glycosylated by galactosylation with ß-glucosidase to give 4'- and 7-ß-galactosides of daidzein, which were new compounds, and α-tocopherol 6-ß-galactoside. These nine glycosides showed higher anti-allergic activity, i.e., inhibitory activity toward histamine release from rat peritoneal mast cells, than their respective aglycones. In addition, these glycosides showed higher tyrosinase inhibitory activity than the corresponding aglycones. Glycosylation of daidzein, α-tocopherol, and hesperetin greatly improved their biological activities.

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.

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 trans-resveratrol by plant-cultured cells.

Plant-cultured cells of Catharanthus roseus converted trans-resveratrol into its 3-O-ß-D-glucopyranoside, 4'-O-ß-D-glucopyranoside, 3-O-(6-O-ß-D-xylopyranosyl)-ß-D-glucopyranoside, and 3-O-(6-O-α-L-arabinopyranosyl)-ß-D-glucopyranoside. The 3-O-(6-O-ß-D-xylopyranosyl)-ß-D-glucopyranoside and 3-O-(6-O-α-L-arabinopyranosyl)-ß-D-glucopyranoside compounds of trans-resveratrol are both new. Incubation of plant-cultured cells of Ipomoea batatas and Strophanthus gratus with trans-resveratrol gave trans-resveratrol 3-O-ß-D-glucopyranoside and trans-resveratrol 4'-O-ß-D-glucopyranoside.

Regioselective glucosidation of trans-resveratrol in Escherichia coli expressing glucosyltransferase from Phytolacca americana.

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-ß-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4'-O-ß-glucoside: 3-O-ß-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.

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.

Effect of Magnesium Ion on the Radical-Scavenging Rate of Pterostilbene in an Aprotic Medium: Mechanistic Insight into the Antioxidative Reaction of Pterostilbene.

Pterostilbene (PTS), a methylated analog of resveratrol (RSV), has recently attracted much attention due to its enhanced bioavailability compared to RSV. However, little is known about the radical-scavenging mechanism of PTS. In this study, we investigated the effect of Mg(ClO4)2 on the scavenging reaction of galvinoxyl radical (GO•) by PTS in acetonitrile (MeCN). GO• was used as a model for reactive oxygen radicals. The second-order rate constant (kH) for the GO•-scavenging reaction by PTS was more than threefold larger than that by RSV, although thermodynamic parameters, such as the relative O-H bond dissociation energies of the phenolic OH groups, ionization potentials, and HOMO energies calculated by the density functional theory are about the same between PTS and RSV. The oxidation peak potential of PTS determined by the cyclic voltammetry in MeCN (0.10 M Bu4NClO4) was also virtually the same as that of RSV. On the other hand, no effect of Mg (ClO4)2 on the kH values was observed for PTS, in contrast to the case for RSV. A kinetic isotope effect of 3.4 was observed when PTS was replaced by a deuterated PTS. These results suggest that a one-step hydrogen-atom transfer from PTS to GO• may be the rate-determining step in MeCN.

Synthesis of xylooligosaccharides of daidzein and their anti-oxidant and anti-allergic activities.

The biocatalytic synthesis of xylooligosaccharides of daidzein was investigated using cultured cells of Catharanthus roseus and Aspergillus sp. ß-xylosidase. The cultured cells of C. roseus converted daidzein into its 4'-O-ß-glucoside, 7-O-ß-glucoside, and 7-O-ß-primeveroside, which was a new compound. The 7-O-ß-primeveroside of daidzein was further xylosylated by Aspergillus sp. ß-xylosidase to daidzein trisaccharide, i.e., 7-O-[6-O-(4-O-(ß-d-xylopyranosyl))-ß-d-xylopyranosyl]-ß-d-glucopyranoside, which was a new compound. The 4'-O-ß-glucoside, 7-O-ß-glucoside, and 7-O-ß-primeveroside of daidzein exerted DPPH free-radical scavenging and superoxide radical scavenging activity. On the other hand, 7-O-ß-glucoside and 7-O-ß-primeveroside of daidzein showed inhibitory effects on IgE antibody production.

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.

Enzymatic synthesis of capsaicin 4-O-ß-xylooligosaccharides by ß-xylosidase from Aspergillus sp.

Capsaicin 4-O-ß-xylooligosaccharides were synthesized by a biocatalytic xylosylation using Aspergillus sp. ß-xylosidase. Capsaicin was converted into three new capsaicin glycosides, i.e. capsaicin 4-O-ß-xyloside, capsaicin 4-O-ß-xylobioside, and capsaicin 4-O-ß-xylotrioside in 15, 12 and 10% yield, respectively. All products were isolated from the reaction mixtures by preparative HPLC. The structures of the products were determined by NMR spectroscopic method.

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.

Synthesis of beta-maltooligosaccharides of glycitein and daidzein and their anti-oxidant and anti-allergic activities.

The production of beta-maltooligosaccharides of glycitein and daidzein using Lactobacillus delbrueckii and cyclodextrin glucanotransferase (CGTase) as biocatalysts was investigated. The cells of L. delbrueckii glucosylated glycitein and daidzein to give their corresponding 4'- and 7-O-beta-glucosides. The beta-glucosides of glycitein and daidzein were converted into the corresponding beta-maltooligosides by CGTase. The 7-O-beta-glucosides of glycitein and daidzein and 7-O-beta-maltoside of glycitein showed inhibitory effects on IgE antibody production. On the other hand, beta-glucosides of glycitein and daidzein exerted 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging activity and supeoxide-radical scavenging activity.

Glycosylation of capsaicinoids with Panax ginseng stimulated by salicylic acid.

The efficient production of ß-glycosides of capsaicin and 8-nordihydrocapsaicin by cultured cells of Panax ginseng is reported. Capsaicin 4-O-(6-O-ß-D-xylopyranosyl)-ß-D-glucopyranoside (ß-primeveroside, 12%) together with capsaicin 4-O-ß-D-glucoside (6%) was isolated from the cell suspension of P. ginseng after one week of incubation with capsaicin. On the other hand, 8-nordihydrocapsaicin was glycosylated to 8-nordihydrocapsaicin 4-O-ß-D-glucoside (5%) and 8-nordihydrocapsaicin 4-O-ß-primeveroside (9%) by P. ginseng. Pretreatment of the cultured cells with salicylic acid greatly enhanced the glucosylation activity toward capsaicinoids. When 500 µM of salicylic acid was added to the cultures prior to the addition of substrate, capsaicin was converted into capsaicin 4-O-ß-D-glucoside (17%) and capsaicin ß-primeveroside (21%) and 8-nordihydrocapsaicin was glycosylated to 8-nordihydrocapsaicin 4-O-ß-D-glucoside (16%) and 8-nordihydrocapsaicin ß-primeveroside (15%).

Relationship between the radical-scavenging activity of selected flavonols and thermodynamic parameters calculated by density functional theory.

The relationship between radical-scavenging rate constants (k) in an aprotic medium and thermodynamic parameters calculated by density functional theory (DFT) was investigated for 7 flavonols, which are myricetin (Myr), quercetin (Que), morin (Mor), kaempferol (Kae), 2'-methylquercetin (2'-MeQue), 5'-methylquercetin (5'-MeQue), and 2',5'-dimethylquercetin (Me2Que). The k values were determined for the reaction between the flavonols and galvinoxyl radical used as a reactivity model of reactive oxygen species in deaerated acetonitrile at 298 K. The energy difference values (D HT, HT: hydrogen transfer) between the flavonols and the corresponding radicals, which equal to the relative O-H bond dissociation energies of the OH groups in the flavonols and ionisation potentials (IP) were calculated by DFT at the B3LYP/6-31++G(d) level with C-PCM solvation model parameterised for acetonitrile. Among the 7 flavonols used in this study, calculated IP values of 4 flavonols exhibited a linear correlation with log k, suggesting that the radical-scavenging reaction of these flavonols may proceed via an electron transfer as the rate determining step.

Glycosylation of sesamol by cultured plant cells.

The glycosylation of sesamol was investigated using cultured cells of Nicotiana tabacum and Eucalyptus perriniana. The cultured suspension cells of N. tabacum converted sesamol into its beta-glucoside (7%) as well as the disaccharide, sesamyl 6-O-(beta-D-glucopyranosyl)-beta-D-glucopyranoside (beta-gentiobioside, 30%). On the other hand, sesamyl 6-O-(alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside (beta-rutinoside, 56%), together with the beta-glucoside (3%), was produced when sesamol was incubated with suspension cells of E. perriniana.

Glycosylation of fluorophenols by plant cell cultures.

Fluoroaromatic compounds are used as agrochemicals and released into environment as pollutants. Glycosylation of 2-, 3-, and 4-fluorophenols using plant cell cultures of Nicotiana tabacum was investigated to elucidate their potential to metabolize these compounds. Cultured N. tabacum cells converted 2-fluorophenol into its beta-glucoside (60%) and beta-gentiobioside (10%). 4-Fluorophenol was also glycosylated to its beta-glucoside (32%) and beta-gentiobioside (6%) by N. tabacum cells. On the other hand, N. tabacum glycosylated 3-fluorophenol to beta-glucoside (17%).

Production of beta-maltooligosaccharides of alpha- and delta-tocopherols by Klebsiella pneumoniae and cyclodextrin glucanotransferase as anti-allergic agents.

The glycosylation of alpha- and delta-tocopherols using Klebsiella pneumoniae and cyclodextrin glucanotransferase (CGTase) was investigated. K. pneumoniae converted alpha- and delta-tocopherols into the corresponding beta-glucosides in 10 and 8% yield, respectively. CGTase glycosylated alpha-tocopheryl beta-glucoside to alpha-tocopheryl beta-maltoside (51%) and alpha-tocopheryl beta-maltotrioside (35%). On the other hand, delta-tocopheryl beta-glucoside was converted into the corresponding beta-maltoside (45%) and beta-maltotrioside (29%) by CGTase. The beta-glucoside of alpha-tocopherol, and beta-glucoside and beta-maltoside of delta-tocopherol showed inhibitory effects on IgE antibody production and on histamine release from rat peritoneal mast cells.

Glycosylation of hesperetin by plant cell cultures.

The biotransformation of hesperetin by cultured cells of Ipomoea batatas and Eucalyptus perriniana was investigated. Three glycosides, hesperetin 3'-O-beta-D-glucopyranoside (33 microg/g fr. wt of cells), hesperetin 3',7-O-beta-D-diglucopyranoside (217 microg/g fr. wt of cells), and hesperetin 7-O-[6-O-(beta-D-glucopyranosyl)]-beta-d-glucopyranoside (beta-gentiobioside, 22 microg/g fr. wt of cells), together with three hitherto known glycosides, hesperetin 5-O-beta-d-glucopyranoside (23 microg/g fr. wt of cells), hesperetin 7-O-beta-D-glucopyranoside (57 microg/g fr. wt of cells), and hesperetin 7-O-[6-O-(alpha-L-rhamnopyranosyl)]-beta-D-glucopyranoside (beta-rutinoside, hesperidin, 13 microg/g fr. wt of cells), were isolated from cultured suspension cells of E. perriniana that had been treated with hesperetin. Oligosaccharide chains were regioselectively formed at the C-7 position of hesperetin to afford beta-gentiobioside and beta-rutinoside. On the other hand, cultured I. batatas cells converted hesperetin into hesperetin 3'-O-beta-D-glucopyranoside (60 microg/g fr. wt of cells), hesperetin 5-O-beta-D-glucopyranoside (23 microg/g fr. wt of cells), and hesperetin 7-O-beta-D-glucopyranoside (110 microg/g fr. wt of cells).