Synthesis of Gentiooligosaccharides of Genistein and Glycitein and Their Radical Scavenging and Anti-Allergic Activity

The synthesis of gentiooligosaccharides of genistein and glycitein using cultured cells of Eucalyptus perriniana as biocatalysts was investigated. The cells of E. perriniana glycosylated genistein and glycitein to give the corresponding 4'-O-β-glucosides, 7-O-β-glucosides, and 7-O-β-gentiobiosides, which were two new compounds. The β-glucosides of genistein and glycitein showed 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging activity and superoxide-radical scavenging activity. On the other hand, 7-O-β-glucosides of genistein and glycitein and the 7-O-β-gentiobioside of glycitein exerted inhibitory effects on IgE antibody production.


Introduction
The soy isoflavonoids genistein and glycitein have been recognized as natural antioxidants, and have been reported to show anti-allergic activities, such as inhibitory effects on histamine release from OPEN ACCESS mast cells [1][2][3][4][5]. However, their use as food-ingredients is limited because of their water-insolubility and low absorbability after oral administration. Glycosylation using cultured cells is useful for preparing water-soluble and stable glycosides from water-insoluble and unstable organic compounds [6][7][8][9][10][11]. For example, it has been reported that glycosylation of the lipophilic flavonoid quercetin improved its absorbability after oral administration [12]. From the viewpoint of pharmacological development of isoflavonoids, their glycosylation is thus of importance. We report herein the biocatalytic glycosylation of genistein and glycitein by cultured cells of Eucalyptus perriniana to produce the corresponding 4'-O-β-glucosides, 7-O-β-glucosides, and 7-O-β-gentiobiosides. We also report their 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging activity, superoxide-radical scavenging activity, and inhibitory activity for IgE antibody formation.

Glycosylation of Genistein (1) and Glycitein (5) by Cultured Cells of E. perriniana
After cultured cells of E. perriniana were incubated with genistein (1) for five days, the glycosylated products 2-4 were isolated from the cells by extraction with MeOH. On the other hand, none were detected in the medium. No additional glycosylation products were detected in the MeOH extracts of the cells despite careful HPLC analyses. On the basis of their HRFABMS, 1 H-and 13 C-NMR, H-H COSY, C-H COSY, and NOE-spectroscopic analyses, the products ( Figure 1) were determined to be genistein 4'-O-β-glucoside (2, 5%), genistein 7-O-β-glucoside (3, 41%), and genistein 7-O-β-gentiobioside (4, 3%), of which 4 is a new compound.
The molecular formula of 4 was established as C 27 H 30 O 15 based on its HRFABMS spectrum, which included a pseudomolecular ion [M + Na] + peak at m/z 617.1488 (calcd. 617.1482 for C 27 H 30 O 15 Na). HRFABMS suggested that 4 was composed of one molecule of 1 and two hexoses. Its 1 H-NMR spectrum showed two anomeric proton signals at δ 4.25 (1H, d, J = 8.0 Hz) and 5.10 (1H, d, J = 7.6 Hz). This suggested the presence of two β-anomers. The 13 C-NMR spectrum included two anomeric carbon signals at δ 99.5 and 102.8. The sugar component of 4 was determined to be β-D-glucopyranose based on the chemical shifts of the carbon signals. The 13 C resonance of C-6'' was shifted downfield to δ 68.6. Correlations were observed between the anomeric proton signal at δ 5.10 (H-1'') and the carbon signal at δ 163.0 (C-7), and between the anomeric proton signal at δ 4.25 (H-1''') and the carbon signal at δ 68.6 (C-6'') in the HMBC spectrum. These findings confirmed that the inner glucopyranosyl residue was attached to the phenolic hydroxyl group at C-7 of genistein (1), and that the pair of β-D-glucopyranosyl residues was 1,6-linked. Thus, 4 was identified as genistein 7-O-[6-O-(β-Dglucopyranosyl)]-β-D-glucopyranoside (7-O-β-gentiobioside).
Next, glycitein (5) was subjected to the same biotransformation system. Glycoside products 6-8 were obtained from the MeOH extracts of the cells. The products were identified as glycitein indicating the presence of two β-anomers in the sugar moiety. The 1 H-and 13 C-NMR spectra of 8 indicated that it was a β-gentiobiosyl analogue of 5. Furthermore, the HMBC spectrum included correlations between the anomeric proton signal at δ 5.12 (H-1'') and the carbon signal at δ 152.5 (C-7), and between the anomeric proton signal at δ 4.50 (H-1''') and the carbon signal at δ 68.9 (C-6''). These findings confirm that the inner β-D-glucopyranosyl residue was attached to the phenolic hydroxyl group at C-7 of glycitein (5) and that the pair of β-D-glucopyranosyl residues were 1,6-linked. Thus,

Radical Scavenging Activity of β-Glycosides of Genistein and Glycitein
The DPPH free-radical scavenging activity of genistein β-glycosides 2-4 and glycitein β-glycosides 6-8 was determined by in vitro bioassay. The antioxidant activities were expressed as IC 50 values and are summarized in Table 1 (7) showed DPPH free-radical scavenging activity. On the other hand, the β-gentiobiosides of genistein and glycitein (4 and 8) had no antioxidant activity. The results obtained here suggested that mono-glucosides of genistein and glycitein would be useful free-radical scavenging antioxidants with aqueous-solubility.
These suggest that formation of long oligosaccharide chains attached to isoflavones might reduce their antioxidant activity. In addition, gentiobioside compounds which contains one phenolic hydroxyl group in their molecule, i.e., salicifolioside A, have been reported to show no DPPH free-radical scavenging activity [14]. It is postulated that gentiobiosylation might eliminate the antioxidant activity

Anti-Allergic Activity of β-Glycosides of Genistein and Glycitein
The effects of genistein β-glycosides 2-4 on IgE antibody formation were examined by an in vivo bioassay using 7S-globulin from soybean as an antigen. The average of rat plasma IgE level after treatment of 7S-globulin with or without test compounds is summarized in Table 2  Next, the average of rat plasma IgE level after treatment of 7S-globulin with or without glycitein β-glycosides 6-8 was examined. Glycitein 7-O-β-D-glucoside (7) and glycitein 7-O-β-gentiobioside (8) showed inhibitory action on IgE antibody formation. The 4'-O-β-D-glucoside (6) of glycitein did not inhibit IgE antibody generation (Table 2).
Recently, we reported that 7-O-β-glycosides of genistein and quercetin showed anti-allergic activity, i.e., suppressive action on histamine release from rat peritoneal mast cells, whereas the β-glycosides, the sugar of which attached at other phenolic hydroxyl groups, exhibited no anti-allergic actions [15].
The results obtained here suggested that β-glucoside and β-gentiobioside at C-7 of genistein and/or glycitein did not attenuate the anti-allergic activity, and that phenolic hydroxyl groups at 4'-position might be necessary for the anti-allergic action of glycosides of genistein and glycitein. Further studies on the mechanism of β-glycosides of genistein and glycitein to act as anti-allergic formulations are now in progress.

General
Genistein and glycotein were purchased from Sigma-Aldrich Co. The NMR spectra were recorded in DMSO-d 6 using a Varian XL-400 spectrometer. The chemical shifts were expressed in δ (ppm) referring to tetramethylsilane. The HRFABMS spectra were measured using a JEOL MStation JMS-700 spectrometer. HPLC was carried out on a YMC-Pack R&D ODS column (150 × 30 mm)

Cultured Cells and Culture Conditions
A cell culture of E. perriniana was induced in our laboratory, and has been cultivated for over 20 years [16]. Cultured E. perriniana cells were subcultured at 4-week intervals on solid Murashige and Skoog (MS) medium (100 mL in a 300-mL conical flask) containing 3% sucrose, 10 mM 2,4-dichlorophenoxyacetic acid, and 1% agar (adjusted to pH 5.7) at 25 °C in the dark. A suspension culture was started by transferring the cultured cells to 100 mL of liquid medium in a 300-mL conical flask, and incubated on a rotary shaker (120 rpm) at 25 °C in the dark. Prior to use for this work, part of the callus tissues (fr. wt 40 g) was transplanted to freshly prepared MS medium (100 mL in a 300-mL conical flask) and grown with continuous shaking for 2 weeks on a rotary shaker (120 rpm).

Production of β-Glycosides of Genistein and Glycitein by E. perriniana
Substrate (0.08 mmol) dissolved in EtOH (300 μL) was individually administered to a 500-mL flask containing suspension cultured cells of E. perriniana. The cultures were then incubated at 25 °C for five days on a rotary shaker (120 rpm) in the dark. After incubation, the cells and medium were separated by filtration with suction. The filtered medium was extracted with EtOAc. The medium was further extracted with n-BuOH. EtOAc and n-BuOH fractions were analyzed by HPLC The cells were extracted with MeOH for 12 h and sonicated for 5 min. The yields of the glycosylation products were calculated on the basis of the peak area from HPLC using calibration curves prepared by HPLC analyses of the authentic glycosides. The MeOH fraction was conc. and partitioned between H 2 O and EtOAc. The

DPPH Radical Scavenging Activity
DPPH free-radical scavenging activities of β-glycosides of genistein and glycotein were determined as follows: DPPH was dissolved in ethanol (500 μM) [17]. The sample solutions were prepared by dissolving each compound in ethanol. To solutions containing various concentrations of each sample (0.1 mL) and ethanol (0.9 mL) was added DPPH solution (1 mL) at room temperature. Vitamin C was used as a positive control. After 20 min at 25 °C, the absorbance was measured at 517 nm. The percentage reduction of the initial DPPH adsorption, i.e., the free-radical scavenging activity, was calculated as follows: E = [(A c − A t ) / A c ] × 100, where A t and A c are the respective absorbance at 517 nm of sample solutions with and without the test compounds. Antioxidant activity was expressed as the 50% inhibitory concentration (IC 50 ).

Superoxide-Radical Scavenging Activity
Superoxide was generated by the xanthine-xanthine oxidase system [17]. Reaction mixture contained 4 mM xanthine (50 μL), various concentration of sample in ethanol (50 μL), 2 mM nitro blue tetrazolium (NBT, 50 μL), 0.3 nkat/mL xanthine oxidase (50 μL), and 0.1 M phosphate buffer (pH 7.4) in a total volume of 2 mL. Vitamin C was used as a positive control. The reaction mixture was incubated at 25 °C for 10 min and the absorbance was read at 560 nm. Percent inhibition was calculated by comparing with control without test compound but containing the same amount of alcohol. The IC 50 value is shown as the sample concentration at which 50% of superoxide-radical was scavenged.